Monocyte-derived Mφs Research Articles

Ezrin drives adaptation of monocytes to the inflamed lung microenvironment

Ezrin, an actin-binding protein, orchestrates the organization of the cortical cytoskeleton and plasma membrane during cell migration, adhesion, and proliferation. Its role in monocytes/macrophages (MΦs) is less understood. Here, we used a monocyte/MΦ-specific ezrin knock-out mouse model to investigate the contribution of ezrin to monocyte recruitment and adaptation to the lung extracellular matrix (ECM) in response to lipopolysaccharide (LPS). Our study revealed that LPS induces ezrin expression in monocytes/MΦs and is essential for monocytes to adhere to lung ECM, proliferate, and differentiate into tissue-resident interstitial MΦs. Mechanistically, the loss of ezrin in monocytes disrupts activation of focal adhesion kinase and AKT serine-threonine protein kinase signaling, essential for lung-recruited monocytes and monocyte-derived MΦs to adhere to the ECM, proliferate, and survive. In summary, our data show that ezrin plays a role beyond structural cellular support, influencing diverse monocytes/MΦ processes and signaling pathways during inflammation, facilitating their differentiation into tissue-resident macrophages.

PARP14 Contributes to the Development of the Tumor-Associated Macrophage Phenotype.

Cancers reprogram macrophages (MΦs) to a tumor-growth-promoting TAM (tumor-associated MΦ) phenotype that is similar to the anti-inflammatory M2 phenotype. Poly(ADP-ribose) polymerase (PARP) enzymes regulate various aspects of MΦ biology, but their role in the development of TAM phenotype has not yet been investigated. Here, we show that the multispectral PARP inhibitor (PARPi) PJ34 and the PARP14 specific inhibitor MCD113 suppress the expression of M2 marker genes in IL-4-polarized primary murine MΦs, in THP-1 monocytic human MΦs, and in primary human monocyte-derived MΦs. MΦs isolated from PARP14 knockout mice showed a limited ability to differentiate to M2 cells. In a murine model of TAM polarization (4T1 breast carcinoma cell supernatant transfer to primary MΦs) and in a human TAM model (spheroids formed from JIMT-1 breast carcinoma cells and THP-1-MΦs), both PARPis and the PARP14 KO phenotype caused weaker TAM polarization. Increased JIMT-1 cell apoptosis in co-culture spheroids treated with PARPis suggested reduced functional TAM reprogramming. Protein profiling arrays identified lipocalin-2, macrophage migration inhibitory factor, and plasminogen activator inhibitor-1 as potential (ADP-ribosyl)ation-dependent mediators of TAM differentiation. Our data suggest that PARP14 inhibition might be a viable anticancer strategy with a potential to boost anticancer immune responses by reprogramming TAMs.

C/EBPα mediates the maturation and antitumor functions of macrophages in human hepatocellular carcinoma

Recent studies have suggested that therapeutic upregulation of CCAAT/enhancer binding protein α (C/EBPα) prevents hepatocellular carcinoma (HCC) progression. However, the mechanisms underlying this outcome are not fully understood. In this study, we investigated the expression and functional roles of C/EBPα in human HCC, with a focus on monocytes/macrophages (Mφs). Paraffin-embedded tissues were used to visualize C/EBPα expression and analyze the prognostic value of C/EBPα+ monocytes/Mφs in HCC patients. The underlying regulatory mechanisms were examined using human monocyte-derived Mφs. The results showed that the expression of C/EBPα on monocytes/Mφs was significantly decreased in intra-tumor tissues compared to the corresponding peri-tumor tissues. C/EBPα+ monocytes/Mφs displayed well-differentiation and antitumor capacities, and the accumulation of these cells in tissue was associated with antitumor immune responses and predicted longer overall survival (OS) of HCC patients. Mechanistic studies demonstrated that C/EBPα was required for Mφ maturation and HLA-DR, CD169 and CD86 expression, which initiates antitumor cytotoxic T-cell responses; however, these effects were inhibited by monocyte autocrine IL-6- and IL-1β-induced suppression of mTOR1 signaling. Reprogramming Mφs via the upregulation of C/EBPα may provide a novel strategy for cancer immunotherapy in patients with HCC.

Abstract 15261: Adipor2 Sirna-Mediated Knockdown Predominantly Polarizes Towards a M1 Phenotype in THP-1 Macrophages

Introduction: Monocyte-derived macrophages (Mϕs) are the major innate immune cells in plaques, where they assume a more pro-inflammatory (M1) or anti-inflammatory (M2) Mϕ phenotype. Notably, M1 Mϕs are more abundant in unstable plaques, while M2 in stable plaques. Adiponectin receptor 1 (AdipoR1) and AdipoR2 are highly expressed in the monocyte/Mϕ lineage and largely mediate immune responses. Herein, we investigated the specific contribution of AdipoR1 and AdipoR2 on polarization towards a M1 or M2 Mϕ phenotype. Methods: siRNA-mediated knockdown of AdipoR1 and/or AdipoR2 was performed in THP-1 monocyte-derived Mϕs, and collected to perform, 1) qRT-PCR analyses on AdipoR1 and AdipoR2, and 2) flow cytometry analyses on M1 Mϕ markers: CD86, CD80, MHC-II, and M2 Mϕ markers: CD206, CD163. Results: For qRT-PCR analyses, siRNA-mediated knockdown of single AdipoR1 and AdipoR2 significantly decreased the mRNA levels of their respective receptor (p<0.01, p<0.0001), while the opposing receptor was unaffected. Double knockdown resulted in a significant decrease in both AdipoR1 and AdipoR2 mRNA levels (p<0.001, p<0.0001). For flow cytometry analyses, single AdipoR1 and double AdipoR1/R2 siRNA led to a significant decrease in ΔMFI (mean fluorescence intensity) of MHC-II (p<0.01), while a significant increase was observed in single AdipoR2 siRNA (p<0.01). Intracellular siRNA-mediated knockdown of AdipoR1 and/or AdipoR2 did not result in a significant loss of their cell surface expression, as confirmed via dot plots analyses. Lastly, single AdipoR1 and double AdipoR1/R2 siRNA exhibited decreased % MHC-II/CD86-positive cells (p<0.01, p<0.001), while single AdipoR2 siRNA increased % MHC-II/CD86-positive cells (p<0.05). Conclusions: Despite intracellular siRNA-mediated knockdown of AdipoR1 and/or AdipoR2, their cell surface expression remains intact. Furthermore, single AdipoR2 siRNA was associated with an increase in M1 Mϕ population (MHC-II/CD86), which is in line with our previous evidence showing decreased AdipoR2 signalling in unstable plaques. Thus, downregulation of AdipoR2 may play a role in polarization predominantly towards a M1 phenotype in THP-1 Mϕs and hence could be mechanistically relevant in the development of unstable plaques.

Human macrophages directly modulate iPSC-derived cardiomyocytes at healthy state and congenital arrhythmia model in vitro.

The electrophysiological regulation of cardiomyocytes (CMs) by the cardiac macrophages (MΦs) has been recently described as an unconventional role of MΦs in the murine heart. Investigating the molecular and physiological modulation of CM by MΦ is critical to understand the novel mechanisms behind cardiac disorders from the systems perspective and to develop new therapeutic approaches. Here, we developed an in vitro direct coculture system to investigate the cellular and functional interaction between human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and monocyte-derived MΦs both in healthy-state and congenital arrhythmia disease model associated with SCN5A ion channel mutations. Congenital arrhythmia patient-derived (P) and healthy individual-derived control (C) monocytes and derived MΦs exhibited distinct M1- and M2-like polarization-related gene expression pattern. The iPSC-CMs and MΦs formed direct membrane contacts in cocultures demonstrated by time-lapse imaging, scanning electron microscopy, and immunolabeling. The intracellular Ca2+ transients were observed in iPSC-CMs and MΦs when in contact with each other. Interestingly, the C-MΦs in direct contact with C-CMs significantly accelerated the contraction rates, demonstrating the positive chronotropic effect of MΦs on healthy cardiac cultures. Furthermore, the MΦs carrying the SCN5A gene mutation significantly enhanced the arrhythmic events in both C-CMs and P-CMs, implying that the sodium channel mutation in the MΦ is important for the CM function. Importantly, when C-MΦs were coupled to tachycardic P-CMs, the contraction frequency drastically decreased, and rhythmicity enhanced implicating the amelioration of the disease phenotype in vitro. Consequently, our results indicated the functional regulatory role of MΦs on human iPSC-CM contractility by membrane contacts in a physiologically relevant in vitro coculture model of both steady-state and arrhythmia. Our findings could serve as a valuable source for the development of effective immunoregulatory therapies for cardiac arrhythmia in the future.

425.6: Recognition of Donor MHCI Disrupts Recipient Reparative Macrophage Differentiation

Introduction: After tissue injury, infiltrating monocytes differentiate into CD86hiLy6chi pro-inflammatory and CD206+CD301+ reparative macrophages (MΦs) that complete repair. MΦs differentiation is coordinated by tissue injury signals, often referred to as damage-associated molecular patterns (DAMPs), and local cytokines. For example, after heart transplant (HTx) IL-33 is released from graft fibroblasts and acts as a reparative/regulatory DAMP that metabolically reprograms MΦs to differentiate into reparative subsets that protect against chronic HTx rejection. Transplanting allogeneic materials is a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses to damaged tissues. For instance, paired immunoglobulin-like receptor (PIR)-A3 on C57BL/6 (B6) monocytes recognition of BALB/c H2-Dd triggers their differentiation into pro-inflammatory dendritic cells that promotes HTx rejection. Monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shapes DAMP-mediated MΦ differentiation after Tx is not understood, so we hypothesize that in addition to DAMPs, recognition of self versus allogeneic MHCI by PIRs modulates reparative or inflammatory MΦ differentiation. Methods: Monocyte-derived MΦ differentiation in response to donor materials was tested by administration of 20x106 irradiated BALB/c allogeneic (H-2d; Allo) or (B6) syngeneic (H-2b; Syn) splenocytes intraperitoneally alone or with recombinant IL-33. Recipients of donor cellular materials were B6 wildtype (WT), Rag2-/-γc-/-, Pira-/- and Pirb-/- mice. The precise impact of H-2Dd (Allo) or H-2Db (Syn) recombinant MHCI on MΦ differentiation was determined using B6 WT and Pira-/- bone marrow-derived MΦs (BMDMs). Flow cytometry was utilized to assess MΦ differentiation. Results: Administration of Syn cells predominantly stimulated monocyte differentiation into CD206+CD301+ reparative MΦs. Allo cells instead drove monocyte differentiation to pro-inflammatory Ly6chiCD86hi MΦs. A similar outcome was observed in Rag2-/-γc-/- recipients, suggesting these changes are not driven by adaptive immunity. Incubation of BMDMs with recombinant allo MHCI, but not syn MHCI, triggered inflammatory Ly6chiCD86hi MΦs in both WT and Pira-/- BMDMs. Co-administration of allo cells with recombinant IL-33 supported differentiation into CD206+CD301+ reparative MΦs. Interestingly, Pirb-/- mice display reduced C206+CD301+ MΦs, which correlated with decreased IL-33 receptor expression. Conclusions: These data indicate allorecognition by monocytes or MΦs disrupts the typical generation of reparative MΦs in response to injury. We show that monocyte-derived MΦs PIR-B recognition of self MHCI may trigger expression of the receptor needed for IL-33-mediated reparative MΦ differentiation. These new data provide important insights into how innate allorecognition and repair pathways intersect to direct the differentiation of reparative or inflammatory MΦs that shape HTx outcomes. #R01AR073527 (HRT) #5T32AI074490-14 (JPW).

Macrophages Derived From Human Induced Pluripotent Stem Cells: The Diversity of Protocols, Future Prospects, and Outstanding Questions.

Macrophages (Mφ) derived from induced pluripotent stem cells (iMphs) represent a novel and promising model for studying human Mφ function and differentiation and developing new therapeutic strategies based on or oriented at Mφs. iMphs have several advantages over the traditionally used human Mφ models, such as immortalized cell lines and monocyte-derived Mφs. The advantages include the possibility of obtaining genetically identical and editable cells in a potentially scalable way. Various applications of iMphs are being developed, and their number is rapidly growing. However, the protocols of iMph differentiation that are currently used vary substantially, which may lead to differences in iMph differentiation trajectories and properties. Standardization of the protocols and identification of minimum required conditions that would allow obtaining iMphs in a large-scale, inexpensive, and clinically suitable mode are needed for future iMph applications. As a first step in this direction, the current review discusses the fundamental basis for the generation of human iMphs, performs a detailed analysis of the generalities and the differences between iMph differentiation protocols currently employed, and discusses the prospects of iMph applications.

Proteomic analysis of exosomes released from Leishmania infected macrophages

Abstract Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani or L. infantum. Parasites reside quiescently in macrophages (Mϕ) but dramatically affect systemic immune responses. These Mϕs, like most eukaryotic cells, release small, protein- and RNA-laden extracellular vesicles called exosomes, which facilitate intercellular communication. We hypothesized exosomes released from infected Mϕs mediate some systemic immune changes in VL. If correct, exoproteomes of infected Mϕs must differ from uninfected Mϕs. Monocyte derived Mϕs (MDMs) from 4 healthy human donors were infected with L. infantum and extracellular parasites removed. Exosomes were collected and exoproteomes were isotope labeled and identified by LC-MS/MS. Data were exported in MaxQuant 1.6.3.3, and analyzed with Perseus 1.6.2.3. After filtering for quality and for proteins found in all 5 technical replicates of at least one group, 244 proteins were quantified. Pairwise comparison revealed significant differences between donors (FDR 0.05, S0=1), and between infected and uninfected states. 2-way ANOVA (p<0.05) revealed 100 proteins differed significantly by infection status, including histone proteins, HSP-70, Prosaposin, Lipoprotein lipase, CD36, Galectin-9 and several histocompatibility regions and antibody constant chains. The data are reflective of dysregulated lipid metabolism known to occur in visceral leishmaniasis. These findings highlight a need to investigate Heat Shock proteins, lipid metabolic pathway proteins, histocompatibility antigens, and histones in extracellular vesicles as potential contributors to the immune dysregulation observed during leishmaniasis.

Different Signaling Pathways Define Different Interferon-Stimulated Gene Expression during Mycobacteria Infection in Macrophages.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) represents one of the greatest threats to human health., Interferons (IFNs) in combination with the first-line of anti-TB drugs have been used for treating TB for decades in the clinic, but how Mtb infection regulates interferon-stimulated genes (ISGs) in human macrophages (Mϕs) remains unknown. In this study, we investigated the expression-signature and associated innate signaling mechanisms of ISGs in Mtb-infected human monocyte-derived Mϕs (hMDMs) and THP-1-derived Mϕs (THP-1-Mϕs). Among 28 of the detected ISGs, 90% of them exerted a significant increase in Mtb-infected Mϕs. Additionally, we found that cytosolic cyclic (GMP-AMP) synthase (cGAS), toll-like receptor-2 (TLR-2) and TLR-4 signaling pathways participated in ISG induction. Their downstream elements of TANK-binding kinase 1 (TBK1), nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase-signal transducer and activator of transcription (JAK-STAT) were selectively involved in Mtb-mediated ISG production. Finally, the numerous types of ISG expression in hMDMs of TB patients were more susceptible to restimulation of Mtb infection or/and IFN treatment than that of healthy people. Hence, different signaling pathways define different ISG expression during Mtb infection and this helps to illustrate how ISGs are elucidated and to better understand the host immune responses to Mtb infection in Mϕs.

P092 Circulating classical monocytes and intestinal macrophages exhibit reduced response to IL-10 in IBD

Mice in which the IL-10 receptor (IL-10R) is knocked out in macrophages (Mφs) alone develop bacterially driven colitis, demonstrating that IL-10 mediated control of these cells is essential to prevent intestinal inflammation. Humans who have loss-of-function IL-10R mutations develop severe early-onset IBD; these individuals may represent the end of a spectrum in which suboptimal control of Mφs by IL-10 leads to gut inflammation. Our aim was to investigate whether monocytes and monocyte-derived intestinal Mφs from adult-onset IBD patients exhibit a diminished response to IL-10. Blood monocyte subsets (CD14++CD16− classical; CD14++CD16+ intermediate; CD14+CD16++ non-classical) and monocyte-derived intestinal Mφs in IBD patients and controls were identified by flow cytometry. Inhibition of LPS-induced TNFα production by IL-10 was measured by intracellular cytokine staining. LPS-induced TNFα production by classical monocytes (78 ± 4.46% TNFα+) was significantly (p < 0.001) inhibited by IL-10 in healthy controls. A similar frequency (89 ± 2.39%) of intermediate monocytes produced TNFα. However, compared with classical monocytes, this response was significantly (p = 0.009) less well controlled by IL-10 despite higher IL-10R expression and similar IL-10-induced STAT3 phosphorylation. Fewer LPS-stimulated non-classical monocytes produced TNFα (33 ± 6.24%; p < 0.001), which was poorly inhibited by IL-10 due to poor IL-10-induced STAT3 phosphorylation as a consequence of low STAT3 availability. IL-10 was significantly less effective at inhibiting TNFα production by classical monocytes from IBD patients than from controls (p = 0.026) (Figure 1), despite increased expression of IL-10Rα and IL-10-induced STAT3 phosphorylation. The implications of a suboptimal response to IL-10 in classical monocytes was investigated in CD14+ monocyte-derived Mφs in the intestine. Two populations of CD45+HLA-DRhi cells were identified based on CD14 expression: CD14hi (P1) and CD14lo (P2). Both populations spontaneously produced TNFα, which was enhanced with LPS stimulation. Inhibition of LPS-induced TNFα by IL-10 was reduced in IBD patients compared with controls. Figure 1. Inhibition of LPS-induced TNFα production in classical monocyte in health and IBD. TNFα production by intermediate and non-classical monocytes is poorly controlled by IL-10 and these populations may contribute to inflammation in the IL-10-rich intestine. A lower responsive to IL-10 observed in both classical monocytes and monocyte-derived intestinal Mφs from IBD patients and may contribute to inflammation.

Effects of IL-10 and Th 2 cytokines on human Mφ phenotype and response to CSF1R inhibitor.

Tumor-associated Mφs display a plastic phenotype that is regulated by the local tumor milieu. Gene expression analysis and functional characterization of Mφs exposed in vitro to individual cytokines aids to delineate the cross-talk between defined cytokines shaping the complex Mφ phenotype. Human monocyte-derived Mφs can be differentiated in vitro with the T helper cell type 2 response cytokines IL-4 and IL-13 or the immunosuppressive IL-10. Notably, only the latter subset undergoes apoptosis when treated with the CSF 1 receptor (CSF1R) blocking antibody emactuzumab. However, under physiologic conditions, the Mφ phenotype is regulated by cytokine combination. Hence, in this study, we characterized the plasticity of IL-4 or IL-13-differentiated Mφs upon exposure to the immunosuppressive IL-10. Although IL-4-differentiated Mφs sustained their molecular phenotype in the presence of IL-10, IL-13-differentiated Mφs were skewed towards the IL-10 phenotype. Gene expression profiling revealed unique IL-4+IL-10 and IL-13+IL-10 Mφ signatures associated with up-regulation of canonical NF-κB or Wnt/β-catenin signaling pathways, respectively. Although IL-10 was able to alter the surface marker and gene expression profile of IL-13-differentiated Mφs, addition of IL-10 did not restore emactuzumab susceptibility. Combining NF-κB and Wnt/β-catenin signaling inhibitors with emactuzumab had no effect on viability. On average 3-5% of cancer patients overexpressed IL-4, IL-13, or IL-10 mRNA in silico. Although a small patient subset overexpressed IL-10+IL-13, IL-4+IL-10 lacked co-expression. In vitro characterization of CSF1R inhibitor-refractory Mφ phenotypes can support novel pharmacological approaches to specifically target these cells.

Macrophage Bactericidal Assays.

The search for the bactericidal activity of macrophage (Mϕ) is crucial not only during infection, but also to explore its functional activities in normal and pathological conditions, such as autoimmune and inflammatory disorders, allergic inflammation, and cancer. There are several methods exploring the phagocytic and bactericidal activities of Mϕ. This chapter focuses specifically on the technique called antibiotic protection assay and on the methods for the determination of Mϕ production of nitric oxide and hydrogen peroxide as antimicrobial agents and biomarkers of respiratory burst. The protocols presented herein are valid for both Mϕ cell lines and monocyte-derived Mϕs (MDMs).

A Discrete Subset of Monocyte-Derived Cells among Typical Conventional Type 2 Dendritic Cells Can Efficiently Cross-Present

Dendritic cells (DCs) and macrophages (Mϕs) share close developmental pathways and functional features, leading to blurring of the boundaries between these two cell lineages. However, a deeper understanding of DC and Mϕ ontogeny and more refined phenotypic and functional characterizations have helped to delineate pre-DC-derived conventional DCs (cDCs), including cDC1s and cDC2s, from monocyte-derived Mϕs. Here, we further refine DC/Mϕ cell classification and report that classically defined cDC2s contain a discrete population ofmonocyte-derived migratory antigen-presenting cells with Mϕ phenotype but functional DC features, including cross-presentation.

CCR2-dependent monocyte-derived macrophages resolve inflammation and restore gut motility in postoperative ileus

ObjectivePostoperative ileus (POI) is assumed to result from myeloid cells infiltrating the intestinal muscularis externa (ME) in patients undergoing abdominal surgery. In the current study, we investigated the role of...

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and β) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus (SA) and Porphyromonas gingivalis (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

Pre/pro-B cells generate macrophage populations during homeostasis and inflammation

Most tissue-resident macrophages (Mφs) are believed to be derived prenatally and are assumed to maintain themselves throughout life by self-proliferation. However, in adult mice we identified a progenitor within bone marrow, early pro-B cell/fraction B, that differentiates into tissue Mφs. These Mφ precursors have non-rearranged B-cell receptor genes and coexpress myeloid (GR1, CD11b, and CD16/32) and lymphoid (B220 and CD19) lineage markers. During steady state, these precursors exit bone marrow, losing Gr1, and enter the systemic circulation, seeding the gastrointestinal system as well as pleural and peritoneal cavities but not the brain. While in these tissues, they acquire a transcriptome identical to embryonically derived tissue-resident Mφs. Similarly, these Mφ precursors also enter sites of inflammation, gaining CD115, F4/80, and CD16/32, and become indistinguishable from blood monocyte-derived Mφs. Thus, we have identified a population of cells within the bone marrow early pro-B cell compartment that possess functional plasticity to differentiate into either tissue-resident or inflammatory Mφs, depending on microenvironmental signals. We propose that these precursors represent an additional source of Mφ populations in adult mice during steady state and inflammation.

Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

Human macrophage polarization enhances opsonophagocytosis and inflammatory response of the stealth pathogen, Treponema pallidum

Human macrophage polarization enhances opsonophagocytosis and inflammatory response of the stealth pathogen, Treponema pallidum

Resolution of acute inflammation bridges the gap between innate and adaptive immunity

AbstractAcute inflammation is traditionally characterized by polymorphonuclear leukocytes (PMN) influx followed by phagocytosing macrophage (Mφs) that clear injurious stimuli leading to resolution and tissue homeostasis. However, using the peritoneal cavity, we found that although innate immune-mediated responses to low-dose zymosan or bacteria resolve within days, these stimuli, but not hyperinflammatory stimuli, trigger a previously overlooked second wave of leukocyte influx into tissues that persists for weeks. These cells comprise distinct populations of tissue-resident Mφs (resMφs), Ly6chi monocyte-derived Mφs (moMφs), monocyte-derived dendritic cells (moDCs), and myeloid-derived suppressor cells (MDSCs). Postresolution mononuclear phagocytes were observed alongside lymph node expansion and increased numbers of blood and peritoneal memory T and B lymphocytes. The resMφs and moMφs triggered FoxP3 expression within CD4 cells, whereas moDCs drive T-cell proliferation. The resMφs preferentially clear apoptotic PMNs and migrate to lymph nodes to bring about their contraction in an inducible nitric oxide synthase-dependent manner. Finally, moMφs remain in tissues for months postresolution, alongside altered numbers of T cells collectively dictating the magnitude of subsequent acute inflammatory reactions. These data challenge the prevailing idea that resolution leads back to homeostasis and asserts that resolution acts as a bridge between innate and adaptive immunity, as well as tissue reprogramming.

Lipopolysaccharide induced expression of pentraxin 3 in human neutrophils and monocyte-derived macrophages

Pentraxin 3 (PTX3) is a prototype protein of long pentraxin. PTX3 is produced by various cells, such as monocytes/macrophages (Mφs) in response to lipopolysaccharide (LPS) and proinflammatory signals. We performed immunoblotting, immunohistochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) of PTX3 in human monocyte-derived Mφs and neutrophils. PTX3 expression was observed in the cytoplasm of both GM-CSF induced monocyte-derived Mφ (GM-Mφ) and M-CSF induced monocyte-derived Mφ (M-Mφ). PTX3 level in both Mφs was up-regulated at 24 h after LPS stimulation. Moreover, we confirmed PTX3 expression in freshly isolated neutrophils, and PTX3 level was distinctly up-regulated at 6 and 24 h after LPS stimulation. These findings suggested that PTX3 expression, not only in Mφs, but also in neutrophils, may reflect the role of PTX3 in inflammation. We believe that PTX3 can contribute as a diagnostic tool to evaluate inflammation at peripheral sites.