Ly6Chi Macrophages Research Articles

Single-cell transcriptomics identifies the differentiation trajectory from inflammatory monocytes to pro-resolving macrophages in a mouse skin allergy model

Both monocytes and macrophages are heterogeneous populations. It was traditionally understood that Ly6Chi classical (inflammatory) monocytes differentiate into pro-inflammatory Ly6Chi macrophages. Accumulating evidence has suggested that Ly6Chi classical monocytes can also differentiate into Ly6Clo pro-resolving macrophages under certain conditions, while their differentiation trajectory remains to be fully elucidated. The present study with scRNA-seq and flow cytometric analyses reveals that Ly6ChiPD-L2lo classical monocytes recruited to the allergic skin lesion sequentially differentiate into Ly6CloPD-L2hi pro-resolving macrophages, via intermediate Ly6ChiPD-L2hi macrophages but not Ly6Clo non-classical monocytes, in an IL-4 receptor-dependent manner. Along the differentiation, classical monocyte-derived macrophages display anti-inflammatory signatures followed by metabolic rewiring concordant with their ability to phagocytose apoptotic neutrophils and allergens, therefore contributing to the resolution of inflammation. The failure in the generation of these pro-resolving macrophages drives the IL-1α-mediated cycle of inflammation with abscess-like accumulation of necrotic neutrophils. Thus, we clarify the stepwise differentiation trajectory from Ly6Chi classical monocytes toward Ly6Clo pro-resolving macrophages that restrain neutrophilic aggravation of skin allergic inflammation.

Protozoan-Derived Cytokine-Transgenic Macrophages Reverse Hepatic Fibrosis.

Macrophage therapy for liver fibrosis is on the cusp of meaningful clinical utility. Due to the heterogeneities of macrophages, it is urgent to develop safer macrophages with a more stable and defined phenotype for the treatment of liver fibrosis. Herein, a new macrophage-based immunotherapy using macrophages stably expressing a pivotal cytokine from Toxoplasma gondii, a parasite that infects ≈ 2 billion people is developed. It is found that Toxoplasma gondii macrophage migration inhibitory factor-transgenic macrophage (Mφtgmif) shows stable fibrinolysis and strong chemotactic capacity. Mφtgmif effectively ameliorates liver fibrosis and deactivates aHSCs by recruiting Ly6Chi macrophages via paracrine CCL2 and polarizing them into the restorative Ly6Clo macrophage through the secretion of CX3CL1. Remarkably, Mφtgmif exhibits even higher chemotactic potential, lower grade of inflammation, and better therapeutic effects than LPS/IFN-γ-treated macrophages, making macrophage-based immune therapy more efficient and safer. Mechanistically, TgMIF promotes CCL2 expression by activating the ERK/HMGB1/NF-κB pathway, and this event is associated with recruiting endogenous macrophages into the fibrosis liver. The findings do not merely identify viable immunotherapy for liver fibrosis but also suggest a therapeutic strategy based on the evolutionarily designed immunomodulator to treat human diseases by modifying the immune microenvironment.

Systemic Mutation of Ncf1 Ameliorates Obstruction-Induced Renal Fibrosis While Macrophage-Rescued NCF1 Further Alleviates Renal Fibrosis.

Aims: NCF1, a subunit of the NADPH oxidase 2 (NOX2), first described the expression in neutrophils and macrophages and participated in the pathogenesis from various systems. However, there are controversial findings on the role of NCF1 in different kinds of kidney diseases. In this study, we aim to pinpoint the specific role of NCF1 in the progression of renal fibrosis induced by obstruction. Results: In this study, NCF1 expression was upregulated in kidney biopsies of chronic kidney disease patients. The expression level of all subunits of the NOX2 complex was also significantly increased in the unilateral ureteral obstruction (UUO) kidney. Then, we used wild-type mice and Ncf1 mutant mice (Ncf1m1j mice) to perform UUO-induced renal fibrosis. Results demonstrated that Ncf1m1j mice exhibited mild renal fibrosis but increased macrophages count and CD11b+Ly6Chi macrophage proportion. Next, we compared the renal fibrosis degree between Ncf1m1j mice and Ncf1 macrophage-rescued mice (Ncf1m1j.Ncf1Tg-CD68 mice). We found that rescuing NCF1 expression in macrophages further alleviated renal fibrosis and decreased macrophage infiltration in the UUO kidney. In addition, flow cytometry data showed fewer CD11b+Ly6Chi macrophages in the kidney of the Ncf1m1j.Ncf1Tg-CD68 group than the Ncf1m1j group. Innovation: We first used the Ncf1m1j mice and Ncf1m1j.Ncf1Tg-CD68 mice to detect the role of NCF1 in the pathological process of renal fibrosis induced by obstruction. Also, we found that NCF1 expressed in different cell types exerts opposing effects on obstructive nephropathy. Conclusion: Taken together, our findings support that systemic mutation of Ncf1 ameliorates renal fibrosis induced by obstruction, and rescuing NCF1 in macrophages further alleviates renal fibrosis.

INFLAMM-AGING IS ASSOCIATED WITH PRO-INFLAMMATORY PROGRAMMING OF INNATE IMMUNE CELLS IN THE COLON

Abstract Chronic low-grade inflammation is prevalent in aging, which is called inflamm-aging. Immune cells are important mediator of inflammatory state of host and the innate immunity is the first responder to various insults. Gastrointestinal track, especially colon, is the site where immune cells are abundant. Aging is associated with increased gut dysbiosis and functional decline. We hypothesize that colonic innate immune cells contribute to the age-associated inflammation in the colon. We found that macrophages in colon mucosa are elevated in aging, and it is accompanied by pro-inflammatory cytokine expression and increased gut permeability. Specifically, we used flow cytometry to assess colonic innate immune cells collected from 5-, 7-, 14-, 19-, 24-, and 28-month-old mice. Aging significantly increased the populations of pro-inflammatory cytokine producing innate immune cells in the colon, including neutrophils, dendritic cells, monocytes and macrophages. Interestingly, the infiltrating Ly6Chi macrophages and pro-inflammatory CD11c+ macrophages were much higher, while anti-inflammatory CD206+ macrophages were decreased in colon of the aged mice. In line with the immune profile, aged mice showed increased gut permeability tested by fluorescein isothiocyanate-dextran, and the gene expressions of gap junction proteins in the colon were decreased, supporting increased gut permeability in the aged mice. Collectively, our results suggest that innate immune cells play an importance role in age-associated gut inflammation; targeting the innate immune cells in the colon may present a novel therapeutic strategy for prevention and treatment of aging leaky gut.

Systemic prime exacerbates the ocular immune response to heat-killed Mycobacterium tuberculosis

Post-infectious uveitis describes the condition of chronic immune mediated ocular inflammation associated with pathogens such as Mycobacterium tuberculosis (Mtb). Mtb associated post-infectious uveitis can be modeled in mice by intravitreal injection of heat-killed Mtb (HKMtb). To better understand how prior systemic exposure to the pathogen alters the local immune response to Mtb, we used flow cytometry and multiplex ELISAs to compare ocular responses to intravitreal HKMtb in the presence or absence of a systemic “prime” of HKMtb. Priming resulted in exacerbation of local inflammation with significantly increased clinical and histologic inflammation scores and increased vitreous cytokines concentrations one day after intravitreal injection of HKMtb. Seven days after injection, uveitis in unprimed animals had largely resolved. In contrast in primed animals, clinical signs of chronic inflammation were associated with a significant increase in the number of ocular T cells, NK cells, and Ly6Chi macrophages and increasing vitreous concentrations of IL-17, VEGF, MIG(CXCL9), IP-10(CXCL10), IL-12p40 and MIP-1α(CCL3). In mice lacking mature T and B cells (RAG2 deficient), the impact of priming on the ocular immune response was ameliorated with significantly lower vitreous cytokine concentrations and spontaneous resolution of uveitis. Altogether these results suggest that the ocular response to Mtb is exacerbated by prior systemic Mtb infection and chronic post-infectious uveitis is mediated by local production of cytokines and chemokines that amplify Th17 and Th1 responses. This mouse model of chronic Mtb associated uveitis will help elucidate mechanisms of disease in patients with post-infectious uveitis.

Early infiltrating macrophage subtype correlates with late-stage phenotypic outcome in a mouse model of hepatorenal fibrocystic disease

Hepatorenal fibrocystic disease (HRFCD) is a genetically inherited disorder related to primary cilia dysfunction in which patients display varying levels of fibrosis, bile duct expansion, and inflammation. In mouse models of HRFCD, the phenotype is greatly impacted by the genetic background in which the mutation is placed. Macrophages are a common factor associated with progression of HRFCD and are also strongly influenced by the genetic background. These data led us to hypothesize that macrophage subtypes that change in relation to the genetic background are responsible for the variable phenotypic outcomes in HRFCD. To test this hypothesis, we utilized a mouse model of HRFCD (Ift88Orpk mice) on the C57BL/6 and BALB/c inbred backgrounds that have well-documented differences in macrophage subtypes. Our analyses of infiltrating macrophage subtypes confirm that genetic strain influences the subtype of infiltrating macrophage present during normal postnatal liver development and in Ift88Orpk livers (Ly6clo in C57BL/6 vs Ly6chi in BALB/c). Each infiltrating macrophage subtype was similarly associated with a unique phenotypic outcome as analysis of liver tissue shows that C57BL/6 Ift88Orpk mice have increased bile duct expansion, but reduced levels of fibrosis compared to BALB/c Ift88Orpk livers. RNA sequencing data suggest that the ability to infiltrate macrophage subtypes to influence the phenotypic outcome may be due to unique ligand-receptor signaling between infiltrating macrophages and cilia dysfunctional biliary epithelium. To evaluate whether specific macrophage subtypes cause the observed phenotypic divergence, we analyzed the liver phenotype in BALB/c Ift88Orpk mice on a CCR2−/− background. Unexpectedly, the loss of Ly6chi macrophages, which were strongly enriched in BALB/c Ift88Orpk mice, did not significantly alter liver fibrosis. These data indicate that macrophage subtypes may correlate with HRFCD phenotypic outcome, but do not directly cause the pathology.In this study, the authors tested the hypothesis that differences in infiltrating macrophage subtypes between C57BL/6 and BALB/c mouse models of hepatorenal fibrocystic disease were responsible for the divergent phenotypic outcome s observed in the liver. Despite the significant correlation between infiltrating macrophage subtype and phenotypic outcome, these correlations were not causative of the liver pathology.

Mesenchymal stem cells attenuate liver fibrosis by targeting Ly6Chi/lo macrophages through activating the cytokine-paracrine and apoptotic pathways

Mesenchymal stem cell (MSC) therapy has become a promising treatment for liver fibrosis due to its predominant immunomodulatory performance in hepatic stellate cell inhibition and fibrosis resolution. However, the cellular and molecular mechanisms underlying these processes remain limited. In the present study, we provide insights into the functional role of bone marrow-derived MSCs (BM-MSCs) in alleviating liver fibrosis by targeting intrahepatic Ly6Chi and Ly6Clo macrophage subsets in a mouse model. Upon chronic injury, the Ly6Chi subset was significantly increased in the inflamed liver. Transplantation of BM-MSCs markedly promoted a phenotypic switch from pro-fibrotic Ly6Chi subset to restorative Ly6Clo subpopulation by secreting paracrine cytokines IL-4 and IL-10 from the BM-MSCs. The Ly6Chi/Ly6Clo subset switch significantly blocked the source of fibrogenic TGF-β, PDGF, TNF-α, and IL-1β cytokines from Ly6Chi macrophages. Unexpectedly, BM-MSCs experienced severe apoptosis and produced substantial apoptotic bodies in the fibrotic liver during the 72 h period of transplantation. Most apoptotic bodies were engulfed by Ly6Clo macrophages, and this engulfment robustly triggered MMP12 expression for fibrosis resolution through the PtdSer-MerTK-ERK signaling pathway. This paper is the first to show previously unrecognized dual regulatory functions of BM-MSCs in attenuating hepatic fibrosis by promoting Ly6Chi/Ly6Clo subset conversion and Ly6Clo macrophage restoration through secreting antifibrogenic-cytokines and activating the apoptotic pathway.

A1 F4/80+LY6CHI MACROPHAGES ARE KEY TO CANCER INITIATION IN COLITIS

Abstract Background Colorectal cancer (CRC) is the third leading cause of cancer death, with a major risk factor being chronic inflammation. Thus, patients with inflammatory bowel disease (IBD) are at an increased risk of CRC. Despite the clear association between inflammation and cancer, the mechanism by which colitis leads to CRC is still not well understood. Aims In this study, we aim to explore the mechanism by which inflammation contributes to the initiation of colitis-associated cancer (CAC). We hypothesize that dextran sodium sulfate (DSS)-induced colitis leads to the infiltration of a specific immune cell type associated with tumorigenesis. Methods Following an injection of azoxymethane (AOM), mice were treated with the colitis-inducing agents DSS, trinitrobenzene sulfonic acid (TNBS), oxazolone (oxa), Citrobacter rodentium, or Doxorubicin (Doxo). The tumor studies were repeated using our published Cre-dependent murine model of CAC. To generate tamoxifen-inducible Cre transgenic mice that allow for Dclk1+ cell lineage tracing and cell-specific knock-out of the tumor suppressor adenomatous polyposis coli (APC), we crossed our Dclk1CreERT2 mice to both ROSA26tdTomato and APCfl/fl mice (Dclk1/APCfl/fl). Results Treatment with DSS, TNBS, oxa, C. rodentium, or Doxo induced colonic inflammation as detected by increased myeloperoxidase (MPO) activity and histologic analysis. DSS administration led to colonic tumors, whereas TNBS, oxa, C. rodentium, or Doxo did not lead to tumorigenesis up to 52 weeks following colitis induction. Upon flow cytometric analysis of several types of immune cells in the colonic tissue, we observed no difference in the number of T and B cells between mice treated with various colitis inducing agents. We did, however, detect significantly increased levels of Ly6G+ neutrophils and F4/80+Ly6Chi macrophages in the DSS-treated mice when compared to mice in the other models of colitis. mRNA and protein array analyses of the colonic tissue, as well as analysis of the RNA-seq data from 206 UC patients (GSE109142), revealed upregulated expression of genes associated with macrophages and neutrophils. Addition of macrophage-produced cytokines, such as IL-1β, TNF-α, or IL-6, induced lineage tracing of Dclk1+ tuft cells in intestinal organoids. Clodronate liposome-mediated depletion of F4/80+Ly6Chi macrophages significantly reduced the number of colonic tumors but did not affect tumor size in Dclk1/APCfl/fl mice. Conclusions Our data suggest that infiltration of F4/80+Ly6Chi macrophages, unique to DSS-induced colitis, leads to colonic tumor formation. This demonstrates that specific immune cell types, rather than the presence of colonic inflammation, plays an important role in the initiation of CAC. Funding Agencies CAG, CIHR

Alternatively activated macrophages promote resolution of necrosis following acute liver injury

Background & AimFollowing acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI).MethodsSeveral phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice.ResultsBMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings.ConclusionWe identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury.Lay summaryAfter an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury.

Matrix metalloproteinase-12 produced by Ly6Clow macrophages prolongs the survival after myocardial infarction by preventing neutrophil influx

BackgroundVarious immune cells are involved in different phases of cardiac repair after myocardial infarction (MI). Especially, Ly6Clow M2-like macrophages (Ly6Clo macrophages) are vital for cardiac repair after MI. However, the molecular mechanisms how Ly6Clo macrophages promote wound healing after MI are still largely unknown. Methods and resultsTranscriptome analysis of Ly6Clo macrophages and Ly6Chigh M1-like macrophages (Ly6Chi macrophages) harvested from the infarcted heart revealed that Ly6Clo macrophages highly expressed matrix metalloproteinase (MMP)-12 mRNA compared to Ly6Chi macrophages. MMP-12 expression was enhanced in the infarcted heart and preferentially observed in Ly6Clo macrophages. Importantly, the survival rate and cardiac function after MI were significantly impaired in MMP-12-deficient (mmp12−/−) mice compared with those in wild-type mice. In addition, the extent of myocardial fibrosis and the number of myofibroblasts in the infarct area were decreased in mmp12−/− mice. MMP-9 expression and neutrophils, which are the major cellular source of MMP-9, in the infarcted heart were increased in mmp12−/− mice. Moreover, mRNA expression of neutrophil-attracting chemokines including CXCL1, CXCL2, and CXCL5 was significantly higher in mmp12−/− mice. Consistently, treatment with anti-CXCR2 antibody significantly decreased neutrophil numbers and MMP-9 expression in the infarcted heart in mmp12−/− mice. Finally, the administration of recombinant MMP-12 into the infarcted heart decreased neutrophil numbers in the infarcted heart and promoted wound healing in both wild-type mice and mmp12−/− mice. ConclusionMMP-12 produced by Ly6Clo macrophages improves the survival after MI possibly through the promotion of wound healing by reducing neutrophil infiltration.

CD24 regulates Ly6Chi macrophages and BDL-induced liver fibrosis

Objective To investigate the regulatory effects of CD24 on Ly6Chi macrophages in liver and its influences on bile duct ligation (BDL)-induced hepatic fibrosis in mice. Methods Liver fibrosis was induced in wild-type (WT) and CD24-/- mice by surgical ligation of the biliary duct. Levels of alanine amino transferase (ALT) in serum were detected and liver sections were stained with haematoxylin and eosin (H&E) staining to assess the severity of liver injury. Sirius Red staining was used to observe the degree of liver fibrosis. Real-time PCR was performed for detecting the expression of hepatic fibrosis-related markers and TGF-β1 at mRNA level. The percentage of macrophages and the number of TGF-β1-producing macrophages were measured by flow cytometry. Results BDL-induced liver fibrosis was exacerbated in CD24-/- mice than in WT mice as demonstrated by more serious hyperplasia in bile duct, more inflammatory infiltration at the portal area and higher levels of ALT in serum. Results of Sirius red staining also showed that the liver fibrosis was more severe in CD24-/- mice than in WT mice. Moreover, α-SMA and collagen typeⅠ alpha 1 (Col1a1) were significantly upregulated in CD24-/- mice. Flow cytometry analysis revealed that CD24 was highly expressed by hepatic macrophages in BDL-induced WT mice, and the percentages of hepatic macrophages were significantly elevated in CD24-/- mice compared with those in WT mice. Further analysis revealed that the percentages of Ly6Chi hepatic macrophages in CD24-/- mice were higher than those in WT mice, but there was no significant difference in the percentages of Ly6Clo macrophages. The expression of TGF-β1 at mRNA level was increased in CD24-/- mice as compared with that in WT mice after BDL. Moreover, intracellular staining showed that Ly6Chi hepatic macrophages in CD24-/- mice secreted more TGF-β1 than the macrophages in WT mice. Conclusions CD24 might attenuate the BDL-induced liver fibrosis in mice via regulating the percentage of hepatic Ly6Chi macrophages and the secretion of TGF-β1. Key words: CD24; Bile duct ligation (BDL); Liver fibrosis; Ly6Chi hepatic macrophage; TGF-β1

Inhibition of FPR2 Impaired Leukocyte Get‐in Signal and Triggers Non‐Resolving Inflammation in Heart Failure

Non‐resolving inflammation is a prominent trigger for heart failure (HF)‐related morbidity and mortality following myocardial infarction (MI). Post‐MI activated leukocyte expressed formyl peptide receptor 2 (FPR2) belongs to the essential for inflammation‐resolution mechanisms. However, the role of FPR2 in leukocyte kinetics in acute HF is incompletely understood. Here, we aimed to determine whether pharmacological inhibition of FPR2 would impair leukocyte trafficking to the site of cardiac injury using WRW4 (FPR2 antagonist). For this, male C57BL/6 (8 to 12 week) mice were subjected to acute HF using permanent coronary artery ligation. FPR2 inhibitor WRW4 (1μg/kg/day; subcutaneous) was injected 3 hours post‐MI and saline‐injected‐mice served as MI‐control. Inhibition of FPR2 impaired leukocyte trafficking (“get‐in” signal) that altered post‐MI immune responsive clearance in the infarcted LV and spleen. Leukocyte kinetics, measured using flow cytometry, showed there was an overall decrease in CD45+/CD11b+; 23.3±2% in WRW4‐injected mice when compared with MI‐control (49.1±2%) in the LV. FPR2 inhibitor, WRW4, decreased FPR2 in the LV and spleen tissue, and mice displayed increase F4/80+/Ly6Chi macrophages (14.8±2%) compared to the MI‐control group (10±1%) in LV, this is indicative of amplified proinflammatory profile. Inhibition of WRW4 primes immature neutrophil infiltration Ly6Gint and intensifies the Ccl2 (all p<0.05) expression compared to MI‐control in the infarcted LV post‐MI. WRW4 increased transcripts of pro‐inflammatory markers TNF‐α and IL‐1β, with a decrease in expression of Arg‐1 (p<0.05) in the infarcted LV compared to MI‐controls is suggestive of non‐resolving or immune suppressive inflammation. In summary, FPR2 inhibition using WRW4 altered leukocyte get‐in signal leading to the onset of frustrated resolution signaling in acute HF post‐MI.Support or Funding InformationHL132989 to GVH and 16POST31000008 to VKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Primary cilia disruption differentially affects the infiltrating and resident macrophage compartment in the liver.

Hepatorenal fibrocystic disease (HRFCD) is characterized by cysts in the kidney and liver with associated fibrosis and is the result of defects in proteins required for cilia function or assembly. Previous reports indicate that macrophages, mainly M2-like macrophages, contribute to HRFCD, although the origin of these cells (yolk sac-derived resident macrophages vs. bone marrow-derived infiltrating macrophages) and their contribution to the observed phenotypes are unknown. We utilize a congenital model of cilia dysfunction (IFT88Orpk) to study the importance of macrophages in HRFCD. Our data show a rapid expansion of the bile duct region and development of fibrosis between 2 and 4 wk of age. Immunofluorescence microscopy analysis reveals an accumulation of F4/80+ macrophages in regions exhibiting biliary hyperplasia in IFT88Orpk mice. Flow cytometry data show that cilia dysfunction leads to an accumulation of infiltrating macrophages (CD11bhi, F4/80lo) and a reduction of resident macrophage (CD11blo, F4/80hi) number. A majority of the infiltrating macrophages are Ly6chi profibrogenic macrophages. Along with the accumulation of immune cells, expression of proinflammatory and profibrotic transcripts, including TGF-β, TNF-α, IL-1β, and chemokine (C-C) motif ligand 2, is increased. Quantitative RT-PCR analysis of flow-sorted cells shows enhanced expression of CCL2 in cholangiocytes and enhanced expression of VEGF-A and IL-6 in Ly6chi macrophages. Genetic inhibition of Ly6chi macrophage accumulation in IFT88Orpk FVB CCR2-/- mice reduced biliary fibrosis but did not affect epithelial expansion. Collectively, these studies suggest that biliary epithelium with defects in primary cilia preferentially recruits Ly6chi infiltrating macrophages, which promote fibrotic progression in HRFCD pathogenesis. NEW & NOTEWORTHY These studies are the first to address the contribution of the infiltrating and resident macrophage niche during progression of hepatorenal fibrocystic disease (HRFCD). We show that the number of infiltrating macrophages is significantly upregulated in HRFCD mouse models. Finally, we show that prevention of Ly6chi infiltrating macrophage accumulation significantly reduces biliary fibrosis, but not biliary hyperplasia, suggesting that this population may be responsible for the fibrotic progression of the disease in HRFCD patients.

The Janus-like Face of IL-4Rα in Macrophages during Liver Fibrosis

The Janus-like Face of IL-4Rα in Macrophages during Liver Fibrosis

Bone marrow-derived cPLA2α contributes to renal fibrosis progression

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme directs a complex "eicosanoid storm" that accompanies the tissue response to injury. cPLA2α and its downstream eicosanoid mediators are also implicated in the pathogenesis of fibrosis in many organs, including the kidney. We aimed to determine the role of cPLA2α in bone marrow-derived cells in a murine model of renal fibrosis, unilateral ureteral obstruction (UUO). WT C57BL/6J mice were irradiated and engrafted with donor bone marrow from either WT mice [WT-bone marrow transplant (BMT)] or mice deficient in cPLA2α (KO-BMT). After full engraftment, mice underwent UUO and kidneys were collected 3, 7, and 14 days after injury. Using picrosirius red, collagen-3, and smooth muscle α actin staining, we determined that renal fibrosis was significantly attenuated in KO-BMT animals as compared with WT-BMT animals. Lipidomic analysis of homogenized kidneys demonstrated a time-dependent upregulation of pro-inflammatory eicosanoids after UUO; KO-BMT animals had lower levels of many of these eicosanoids. KO-BMT animals also had fewer infiltrating pro-inflammatory CD45+CD11b+Ly6Chi macrophages and reduced message levels of pro-inflammatory cytokines. Our results indicate that cPLA2α and/or its downstream mediators, produced by bone marrow-derived cells, play a major role in eicosanoid production after renal injury and in renal fibrinogenesis.

Infiltrating macrophages are broadly activated at the early stage to support acute skeletal muscle injury repair

Acute skeletal muscle injury repair requires an adequate inflammatory response predominated by macrophage infiltration. We studied the activation of infiltrating macrophages by analyzing the expression of M1/M2 signature genes. Most of the intramuscular macrophages were Ly6Chi at day 1 after BaCl2 injection, while many were Ly6Clo at day 3. Ly6Chi macrophages at day 1 expressed a high level of both M1 and M2 genes, and the Ly6Chi and Ly6Clo macrophages at day 3 expressed a similar level of many M1/M2 genes. Infiltrating macrophages are broadly activated rather than polarized at the early stage to support acute skeletal muscle injury repair.

The dendritic cell-T helper 17-macrophage axis controls cholangiocyte injury and disease progression in murine and human biliary atresia.

These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188).

Choroidal Neovascularization Is Inhibited in Splenic-Denervated or Splenectomized Mice with a Concomitant Decrease in Intraocular Macrophage.

PurposeTo determine the involvement of sympathetic activity in choroidal neovascularization (CNV) using laser-induced CNV in a mouse model.MethodsWe investigated changes in the proportions of intraocular lymphocytes, granulocytes, and three macrophage subtypes (Ly6Chi, Ly6Cint, and Ly6Clo) after laser injury in mice using flow cytometry, and evaluated CNV lesion size in mice lacking inflammatory cells. Further, we evaluated the lesion size in mice administered the β3 receptor antagonist, splenic-denervated and splenectomized mice. We also assessed changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenic-denervated and splenectomized mice. Lastly, lesion size was compared between splenic-denervated mice with or without adoptive transfer of macrophages following laser injury. After Ly5.1 mice spleen-derived Ly6Chi cells were transferred into Ly5.2 mice, the proportions of intraocular Ly5.1+Ly6Chi cells were compared.ResultsIn WT mice, the proportion of CD4+ T cells recruited into the eye increased progressively from day 3 to day 7 after laser injury, whereas, intraocular CD8+ T cells did not change significantly. Proportions of B220+ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6Chi and Ly6Clo) peaked at day 3 following laser injury. In contrast, Ly6Cint/loCD64+ subtype showed a significantly higher percentage at day 7 after laser injury. There were no differences in lesion size between CD4–/–or Rag2–/–mice and controls, whereas lesion size was significantly reduced in CCR2−/− mice and clodronate liposome-treated mice. CNV lesion area was significantly reduced in mice with β3 blocker treatment, splenic-denervated and splenectomized mice compared with controls. Intraocular Ly6Chi macrophages were also reduced by splenic denervation or splenectomy. Adoptive transfer of spleen-derived Ly6Chi cells increased the lesion size in splenic-denervated mice. Compared with controls, intraocular donor-derived Ly6Chi cells recruited into the eye were reduced in splenic-denervated and splenectomized mice.ConclusionsAlthough lymphocytes had little effect on CNV formation, Ly6Chi macrophages/monocytes exacerbated CNV in mice. Sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye.

The spleen as an extramedullary source of inflammatory cells responding to acetaminophen-induced liver injury

Macrophages have been shown to play a role in acetaminophen (APAP)-induced hepatotoxicity, contributing to both pro- and anti-inflammatory processes. In these studies, we analyzed the role of the spleen as an extramedullary source of hepatic macrophages. APAP administration (300mg/kg, i.p.) to control mice resulted in an increase in CD11b+ infiltrating Ly6G+ granulocytic and Ly6G− monocytic cells in the spleen and the liver. The majority of the Ly6G+ cells were also positive for the monocyte/macrophage activation marker, Ly6C, suggesting a myeloid derived suppressor cell (MDSC) phenotype. By comparison, Ly6G− cells consisted of 3 subpopulations expressing high, intermediate, and low levels of Ly6C. Splenectomy was associated with increases in mature (F4/80+) and immature (F4/80−) pro-inflammatory Ly6Chi macrophages and mature anti-inflammatory (Ly6Clo) macrophages in the liver after APAP; increases in MDSCs were also noted in the livers of splenectomized (SPX) mice after APAP. This was associated with increases in APAP-induced expression of chemokine receptors regulating pro-inflammatory (CCR2) and anti-inflammatory (CX3CR1) macrophage trafficking. In contrast, APAP-induced increases in pro-inflammatory galectin-3+ macrophages were blunted in livers of SPX mice relative to control mice, along with hepatic expression of TNF-α, as well as the anti-inflammatory macrophage markers, FIZZ-1 and YM-1. These data demonstrate that multiple subpopulations of pro- and anti-inflammatory cells respond to APAP-induced injury, and that these cells originate from distinct hematopoietic reservoirs.

6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6chi Ly6g− monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6clo Ly6g+ granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6chi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8+ T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1702-8) contains supplementary material, which is available to authorized users.