Tissue-resident Macrophages Research Articles

Macrophage Fate Mapping.

Tissue‐resident macrophages are present in all tissues where they perform homeostatic and immune surveillance functions. In many tissues, resident macrophages develop from embryonic progenitors, which mature into a self‐maintaining population through local proliferation. However, tissue‐resident macrophages can be supported by recruited monocyte‐derived macrophages during scenarios such as tissue growth, infection, or sterile inflammation. Circulating blood monocytes arise from hematopoietic stem cell progenitors and possess unique gene profiles that support additional functions within the tissue. Determining cell origins (ontogeny) and cellular turnover within tissues has become important to understanding monocyte and macrophage contributions to tissue homeostasis and disease. Fate mapping, or lineage tracing, is a promising approach to tracking cells based on unique gene expression driving reporter systems, often downstream of a Cre‐recombinase–mediated excision event, to express a fluorescent protein. This approach is typically deployed temporally with developmental stage, disease onset, or in association with key stages of inflammation resolution. Importantly, myeloid fate mapping can be combined with many emerging technologies, including single‐cell RNA‐sequencing and spatial imaging. The application of myeloid cell fate mapping approaches has allowed for impactful discoveries regarding myeloid ontogeny, tissue residency, and monocyte fate within disease models. This protocol outline will discuss a variety of myeloid fate mapping approaches, including constitutive and inducible labeling approaches in adult and embryo tissues. This article outlines basic approaches and models used in mice for fate mapping macrophages. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Adult Fate Mapping Basic Protocol 2: Embryonic Fate Mapping

Diversity and function of brain-associated macrophages.

Different tissue-resident macrophages are present in and at the borders of the brain. Microglia reside in the central nervous systemparenchyma, whereas non-parenchymal macrophages dwell at the interface between the brain and the periphery, including the perivascular spaces, the choroid plexus, and the meninges. Microglia display regional heterogeneity in phenotype and function, and fulfill critical roles in neuronal development and regulation of brain homeostasis in the healthy brain. In recent years, it became clear that the non-parenchymal macrophages also exhibit transcriptional diversity and different dynamics but less is known about their functional specification at their respective anatomical locations. This review discusses recent advances describing the heterogeneity of the brain macrophage compartment and potential physiological functions with a focus on non-parenchymal macrophages.

RXRα Regulates the Development of Resident Tissue Macrophages.

Resident tissue macrophages (RTMs) develop from distinct waves of embryonic progenitor cells that seed tissues before birth. Tissue-specific signals drive a differentiation program that leads to the functional specialization of RTM subsets. Genetic programs that regulate the development of RTMs are incompletely understood, as are the mechanisms that enable their maintenance in adulthood. In this study, we show that the ligand-activated nuclear hormone receptor, retinoid X receptor (RXR)α, is a key regulator of murine RTM development. Deletion of RXRα in hematopoietic precursors severely curtailed RTM populations in adult tissues, including the spleen, peritoneal cavity, lung, and liver. The deficiency could be traced to the embryonic period, and mice lacking RXRα in hematopoietic lineages had greatly reduced numbers of yolk sac and fetal liver macrophages, a paucity that persisted into the immediate postnatal period.

MiR-146a Maintains Immune Tolerance of Kupffer Cells and Facilitates Hepatitis B Virus Persistence in Mice.

Kupffer cells (KCs), the largest tissue-resident macrophage population in the body, play a central role in maintaining a delicate balance between immune tolerance and immunity in the liver. However, the underlying molecular mechanism remains elusive. In this study, we show that KCs express high levels of miR-146a, which is under control of the PU.1 transcription factor. miR-146a deficiency promoted KCs differentiation toward a proinflammatory phenotype; conversely, miR-146a overexpression suppressed this phenotypic differentiation. We found that hepatitis B virus (HBV) persistence or HBV surface Ag treatment significantly upregulated miR-146a expression and thereby impaired polarization of KCs toward a proinflammatory phenotype. Furthermore, in an HBV carrier mouse model, KCs depletion by clodronate liposomes dramatically promoted HBV clearance and enhanced an HBV-specific hepatic CD8+ T cell and CD4+ T cell response. Consistent with this finding, miR-146a knockout mice cleared HBV faster and elicited a stronger adaptive antiviral immunity than wild-type mice. In vivo IL-12 blockade promoted HBV persistence and tempered the HBV-specific CTL response in the liver of miR-146a knockout mice. Taken together, our results identified miR-146a as a critical intrinsic regulator of an immunosuppressive phenotype in KCs under inflammatory stimuli, which may be beneficial in maintenance of liver homeostasis under physiological condition. Meanwhile, during HBV infection, miR-146a contributed to viral persistence by inhibiting KCs proinflammatory polarization, highlighting its potential as a therapeutic target in HBV infection.

IL-10 and TGF-β Increase Connexin-43 Expression and Membrane Potential of HL-1 Cardiomyocytes Coupled with RAW 264.7 Macrophages.

Cardiac resident macrophages facilitate electrical conduction by interacting with cardiomyocytes via connexin-43 (Cx43) hemichannels. Cx43 is critical for impulse propagation and coordination between muscle contractions. Cardiomyocyte electrophysiology can be altered when coupled with noncardiomyocyte cell types such as M2c tissue-resident macrophages. Using cocultures of murine HL-1 cardiomyocytes and RAW 264.7 macrophages, we examined the hypothesis that cytokine signals, TGF-β1 and IL-10, upregulate Cx43 expression at points of contact between the two cell types. These cytokine signals maintain the macrophages in an M2c anti-inflammatory phenotype, mimicking cardiac resident macrophages. The electrophysiology of cardiomyocytes was examined using di-8-ANEPPS potentiometric dye, which reflects a change in membrane potential. Greater fluorescence intensity of di-8-ANEPPS occurred in areas where macrophages interacted with cardiomyocytes. Suppressor of cytokine signaling 3 (SOCS3) peptide mimetic downregulated fluorescence of this membrane potentiometric stain. Cx43 expression in cocultures was confirmed by fluorescence microscopy and flow cytometry. Confocal images of these interactions demonstrate the Cx43 hemichannel linkages between the cardiomyocytes and macrophages. These results suggest that TGF-β1 and IL-10 upregulate Cx43 hemichannels, thus enhancing macrophage-cardiomyocyte coupling, raising the cellular resting membrane potential and leading to a more excitatory cardiomyocyte.

The Role of Tissue-Resident Macrophages in the Development and Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), comprising Crohn’s disease and ulcerative colitis, is a refractory disease with many immune abnormalities and pathologies in the gastrointestinal tract. Because macrophages can distinguish innocuous antigens from potential pathogens to maintain mucosa barrier functions, they are essential cells in the intestinal immune system. With numerous numbers in the intestinal tract, tissue-resident macrophages have a significant effect on the constant regeneration of intestinal epithelial cells and maintaining the immune homeostasis of the intestinal mucosa. They also have a significant influence on IBD through regulating pro-(M1) or anti-inflammatory (M2) phenotype polarization according to different environmental cues. The disequilibrium of the phenotypes and functions of macrophages, disturbed by intracellular or extracellular stimuli, influences the progression of disease. Further investigation of macrophages’ role in the progression of IBD will facilitate deciphering the pathogenesis of disease and exploring novel targets to develop novel medications. In this review, we shed light on the origin and maintenance of intestinal macrophages, as well as the role of macrophages in the occurrence and development of IBD. In addition, we summarize the interaction between gut microbiota and intestinal macrophages, and the role of the macrophage-derived exosome. Furthermore, we discuss the molecular and cellular mechanisms participating in the polarization and functions of gut macrophages, the potential targeted strategies, and current clinical trials for IBD.

Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection.

While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

HIV Latency in Myeloid Cells: Challenges for a Cure.

The use of antiretroviral therapy (ART) for Human Immunodeficiency Virus (HIV) treatment has been highly successful in controlling plasma viremia to undetectable levels. However, a complete cure for HIV is hindered by the presence of replication-competent HIV, integrated in the host genome, that can persist long term in a resting state called viral latency. Resting memory CD4+ T cells are considered the biggest reservoir of persistent HIV infection and are often studied exclusively as the main target for an HIV cure. However, other cell types, such as circulating monocytes and tissue-resident macrophages, can harbor integrated, replication-competent HIV. To develop a cure for HIV, focus is needed not only on the T cell compartment, but also on these myeloid reservoirs of persistent HIV infection. In this review, we summarize their importance when designing HIV cure strategies and challenges associated to their identification and specific targeting by the “shock and kill” approach.

Digital Cell Atlas of Mouse Uterus: From Regenerative Stage to Maturational Stage.

Endometrium undergoes repeated repair and regeneration during the menstrual cycle. Previous attempts using gene expression data to define the menstrual cycle failed to come to an agreement. Here we used single-cell RNA sequencing data of C57BL/6J mice uteri to construct a novel integrated cell atlas of mice uteri from the regenerative endometrium to the maturational endometrium at the single-cell level, providing a more accurate cytological-based elucidation for the changes that occurred in the endometrium during the estrus cycle. Based on the expression levels of proliferating cell nuclear antigen, differentially expressed genes, and gene ontology terms, we delineated in detail the transitions of epithelial cells, stromal cells, and immune cells that happened during the estrus cycle. The transcription factors that shaped the differentiation of the mononuclear phagocyte system had been proposed, being Mafb, Irf7, and Nr4a1. The amounts and functions of immune cells varied sharply in two stages, especially NK cells and macrophages. We also found putative uterus tissue-resident macrophages and identified potential endometrial mesenchymal stem cells (high expression of Cd34, Pdgfrb, Aldh1a2) in vivo. The cell atlas of mice uteri presented here would improve our understanding of the transitions that occurred in the endometrium from the regenerative endometrium to the maturational endometrium. With the assistance of a normal cell atlas as a reference, we may identify morphologically unaffected abnormalities in future clinical practice. Cautions would be needed when adopting our conclusions, for the limited number of mice that participated in this study may affect the strength of our conclusions.

Epigenetic regulation of innate immune memory in microglia

BackgroundMicroglia are the tissue-resident macrophages of the CNS. They originate in the yolk sac, colonize the CNS during embryonic development and form a self-sustaining population with limited turnover. A consequence of their relative slow turnover is that microglia can serve as a long-term memory for inflammatory or neurodegenerative events.MethodsUsing ATAC-, ChIP- and RNA-sequencing, we characterized the epigenomes and transcriptomes of FACS-purified microglia from mice exposed to different stimuli. A repeated endotoxin challenge (LPS) was used to induce tolerance in microglia, while genotoxic stress (DNA repair deficiency-induced accelerated aging through Ercc1 deficiency) resulted in primed (hypersensitive) microglia.ResultsWhereas the enrichment of permissive epigenetic marks at enhancer regions could explain training (hyper-responsiveness) of primed microglia to an LPS challenge, the tolerized response of microglia seems to be regulated by loss of permissive epigenetic marks. We identify that inflammatory stimuli and accelerated aging as a result of genotoxic stress activate distinct gene networks. These gene networks and associated biological processes are partially overlapping, which is likely driven by specific transcription factor networks, resulting in altered epigenetic signatures and distinct functional (desensitized vs. primed) microglia phenotypes.ConclusionThis study provides insight into epigenetic profiles and transcription factor networks associated with transcriptional signatures of tolerized and trained microglia in vivo, leading to a better understanding of innate immune memory of microglia.

Weathering the Storm: Harnessing the Resolution of Inflammation to Limit COVID-19 Pathogenesis.

The resolution of inflammation is a temporally and spatially coordinated process that in its innate manifestations, primarily involves neutrophils and macrophages. The shutdown of infection or injury-induced acute inflammation requires termination of neutrophil accumulation within the affected sites, neutrophil demise, and clearance by phagocytes (efferocytosis), such as tissue-resident and monocyte-derived macrophages. This must be followed by macrophage reprogramming from the inflammatory to reparative and consequently resolution-promoting phenotypes and the production of resolution-promoting lipid and protein mediators that limit responses in various cell types and promote tissue repair and return to homeostatic architecture and function. Recent studies suggest that these events, and macrophage reprogramming to pro-resolving phenotypes in particular, are not only important in the acute setting, but might be paramount in limiting chronic inflammation, autoimmunity, and various uncontrolled cytokine-driven pathologies. The SARS-CoV-2 (COVID-19) pandemic has caused a worldwide health and economic crisis. Severe COVID-19 cases that lead to high morbidity are tightly associated with an exuberant cytokine storm that seems to trigger shock-like pathologies, leading to vascular and multiorgan failures. In other cases, the cytokine storm can lead to diffuse alveolar damage that results in acute respiratory distress syndrome (ARDS) and lung failure. Here, we address recent advances on effectors in the resolution of inflammation and discuss how pro-resolution mechanisms with particular emphasis on macrophage reprogramming, might be harnessed to limit the universal COVID-19 health threat.

The contribution of fetal-derived tissue resident macrophages to cytomegalovirus-associated sensorineural hearing loss

Abstract Cytomegalovirus (CMV) is the most common congenital viral infection in the developed world and the leading cause of non-genetic sensorineural hearing loss (SNHL) in children. Congenital CMV causes progressive SNHL long after acute infection has resolved, suggesting a lasting effect on the developing host immune response within the cochlea and surrounding temporal bone marrow (TB). Using fate-mapping models, we have investigated the contribution of fetal-derived tissue resident macrophages (TRMs) to normal cochlear development and function, and their role in mediating CMV-associated SNHL. Our analysis defined two distinct myeloid population within the inner ear: 1) classical tissue resident macrophages (F4/80hiCD11blo) that highly express CD64 and are found exclusively in cochlear tissue; and 2) transitional monocytes (F4/80midCD11bhi) that express both CD64 and Ly6C and are found in cochlea and TB. Fate mapping of these populations uncovered dual contribution of fetal-derived cochlear TRMs from yolk sac (YS) and fetal liver (FL) hematopoiesis. Transitional monocytes increase during acute CMV infection, acting as drivers of expanded lymphoid populations after CMV resolves. Importantly, analysis of Flt3-Cre labeling within myeloid populations suggests that TRMs are replaced by BM-derived populations at later postnatal stages following CMV infection, coinciding with SNHL. Ongoing work examines spatial distribution of distinct fetal-derived TRMs along cochlear architecture, defining transcriptional regulation differences between YS-, FL-, and adult-derived TRMs in response to CMV, and examining how replacement of fetal-derived TRMs with adult-derived TRMs affects tissue function and response to infection. Supported by grants from University of Utah Molecular Medicine and Immunology, Inflammation and Infectious Disease (3i) Initiative and NIAID (T32 5T32AI138945)

Single-cell multi-omic analysis identify heterogeneity and distinct features in fate-mapped tissue-resident alternatively activated macrophages

Abstract Peritoneal cavity cells play pivotal roles in inflammation, repair, and maintaining homeostasis in response to pathogenic infections and tissue injury. Two major tissue-resident macrophages (TRMs) are large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). These are the major macrophage subsets in the peritoneal cavity and originate from embryogenic (LPMs) or bone-marrow-derived myeloid precursors (SPMs). CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) provides simultaneous information for single cells in both cell-surface protein and gene expression levels. Here, we used CITE-seq to profile peritoneal cells by an oligonucleotide-labeled antibody panel designed to react with 189 unique mouse cell surface antigens. We identify 14 markers exclusively expressed on TRMs but not other immune cell types. These markers can classify phenotype differences between LPMs and SPMs during IL-4 stimulation. We further profile fate-mapped TRMs by scRNA-seq and identified more heterogenous phenotypes of TRMs that originated from embryogenic rather than bone-marrow-derived myeloid precursors. Notably, serum amyloid A-3 (Saa3) and platelet factor 4 (Pf4), can distinguish TRM clusters from alternative activation to IL-4 stimulation and Heligmosomoides polygyrus infection. Hence, we identify distinct markers that can be used to distinguish the different origins and heterogenous TRM phenotypes under steady state and type 2 immune responses.

Role of CD73 on peritoneal macrophages in age-associated immune dysfunction and tumor immunity

Abstract Aging is a major risk factor for cancer and many other life-threatening diseases due in part to a wide range of age-associated immune dysfunctions. In the context of cancer, chronic, low-grade inflammation seen with aging is thought to contribute to cellular transformation and tumorigenesis. Tissue resident macrophages (TRM) are key innate immune regulators of the inflammatory milieu in all tissues, and studies from our laboratory have shown that expression of the 5’-ectonucleotidase CD73 on macrophages suppresses inflammatory responses and promotes the resolution of tissue inflammation in the lung and peritoneum. However, the role of CD73 on TRM in modulating inflammation during aging or how age-associated changes in CD73 on TRM contribute to cancer development are not known. We have found that large peritoneal macrophages from aged mice (18+ months) show significantly increased levels of surface CD73 compared to young mice (<4 months). To test the role of CD73 in regulating inflammation in vivo, we utilized mouse models of ovarian cancer and peritonitis. Our preliminary studies suggest that blocking CD73 in aged mice reduces LPS-mediated production of TNF-a in vivo and ex vivo when compared to young mice. In addition, we found that treatment of ID8-Def29/Vegf-a tumor-bearing mice with anti-CD73 monoclonal antibody significantly prolongs the survival of aged mice compared to young mice. In ongoing experiments, we are utilizing conditional deletion approaches to address the role of CD73 specifically on macrophages in these models. Results from this study will provide insight into the immunoregulatory role of CD73 on TRM during aging and cancer, and will help refine therapeutic approaches targeting CD73 in cancer immunotherapy. Supported by NIH grants T32 AI007496 and R01 AI114554.

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ Tcells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

Ovariectomy‐Induced Arterial Stiffening is Associated with Downregulation of Tissue Resident Macrophage Markers

Tissue resident macrophages play a major role in the regulation of the extracellular matrix (ECM). The ECM provides an essential scaffold and mechanical stability for blood vessels, but remodeling due to acute injury or chronic inflammatory conditions like hypertension promotes vascular stiffening. We have previously shown that pulse wave velocity (PWV), a measure of arterial stiffness, is higher in male and ovariectomized (OVX) female mice versus intact female mice. RNA‐sequencing of aortas from intact and OVX mice revealed changes in several genes involved in fibrosis as well as macrophage function. Therefore, we hypothesized that estrogen protects from arterial stiffening through maintenance of “protective” macrophages and limiting pro‐fibrotic ECM components. Based on the RNAseq results, we assessed the impact of estrogen on aortic expression of four fibrosis‐associated genes: decorin (DCN), endothelin‐1 (Edn1), type III collagen α1 (Col3a1), and connective tissue growth factor (CTGF) as well as four cell surface macrophage markers: colony stimulating factor 1 receptor (CSF1R), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), AXL receptor tyrosine kinase (Axl), and growth arrest specific 6 (Gas6).MethodsFemale C57BL/6 mice were ovariectomized (OVX) at 8 weeks or remained intact. PWV was obtained as a measure of arterial stiffness using a Vevo 1100 high resolution ultrasound. Mice were euthanized between 20‐23 weeks of age for tissue collection. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using an unpaired t‐test.ResultsAbdominal PWV was increased in OVX mice compared with intact mice (p<0.0001). Surprisingly, OVX induced a significant decrease (p=0.045) in DCN expression, while a downward trend was found for CTGF (p=0.10) and Col3a1 (p=0.08) but not Edn1 (p=0.59). OVX also decreased the expression of macrophage markers CSF1R (p=0.022) and LYVE1 (p=0.047), with a downward trend for Gas6 (p=0.096) but not Axl (0.161).ConclusionIncreased PWV in OVX mice was not accompanied by upregulation of pro‐fibrotic genes but was associated with decreases in macrophage markers CSF1R and LYVE1 in the aorta. Therefore, estrogen loss may promote vascular stiffening through changes in the amounts or functions of specific subsets of macrophages in the vasculature. Our data support previous studies establishing a relationship between CSF1R, LYVE1, and arterial homeostasis. Future studies will examine the impact of estrogen on aortic tissue resident macrophages using flow cytometry and the downstream signaling that promotes arterial stiffening.

Epithelial Sodium Channels in Monocyte‐Macrophage Migration and Regulation by Pro‐inflammatory Cytokines TNFα and IFNγ

Migration of monocytes‐macrophages play an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. While Epithelial Na+ Channels (ENaC) are required for normal migratory responses in glial, trophoblast, and vascular smooth muscle cells, their role in monocyte‐macrophage migration signaling is unknown. The Human Protein Atlas data base (www.proteinatlas.org) shows ENaC message in peripheral blood mononuclear cells and tissue resident macrophages in human populations. We used an in vitro model to determine the importance of ENaC in chemotactic migration of monocytes‐macrophages using a standard Boyden chamber assay. Cells were suspended in 0.4% FBS ± amiloride (0.1‐10 µM) or benzamil (0.01‐1 µM) to inhibit ENaC channels, then migrated for 4 hr towards a 10% FBS gradient. In mouse RAW cells, migration responses were inhibited by amiloride (65±6 – 46±4% of control at 0.1 and 10 µM, p=0.0001) and benzamil (55±6 – 55±5% of control at 0.01 and 1.0 µM, p<0.0001). In human THP‐1 cells, migration responses were similarly inhibited by amiloride (73±5 ‐ 40±6% of control at 0.1 and 10 µM, p<0.0001) and benzamil (68±7 – 53±4% of control at 0.01 and 1.0 µM, p<0.0001). Since benzamil and amiloride are selective for ENaC at sub and low micromolar concentrations, respectively, our findings indicate ENaC signaling is required for migration of unstimulated monocyte‐macrophage cells. To determine if pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interferon gamma (INFγ), regulate ENaC expression and function, we used TaqMan quantitative PCR, western blotting and chemotactic migration. Cells were exposed to a standard protocol to activate macrophages: INFγ for 48 hr plus TNFα (10 ng/mL each) during the last 24 hr. Expression of α and βENaC and GAPDH was calculated as fold change using the delta delta CT method (2‐∆∆CT). INFγ48hr + TNFα24hr increased α and βENaC message (9±2 and 5±1, n=4‐6, p=0.01). γENaC was undetectable. Despite increased message, whole cell αENaC protein expression was inhibited 50% by western blotting. The mechanism underlying message stimulation and protein inhibition remains to be determined. A similar effect on message upregulation and migration inhibition also occurred with 18 hr INFγ or TNFα alone (0‐10 ng/mL). Activation with INFγ/TNFα inhibited migration up to 70% and abolished the amiloride (1 µM) sensitive component. In unstimulated cells, amiloride (1 µM) coculture induced a rebound increase in migration following withdrawal, which was abolished by INFγ/TNFα treatment. This finding suggests cytokine induced ENaC loss mediates the migration inhibition. In summary, ENaC mediates monocyte migration, however, monocyte activation by INFγ/TNFα inhibits migration by inhibiting ENaC. Since pro‐inflammatory cytokines initiate monocyte differentiation and polarization to the phagocytic M1 phenotype, future studies will determine if ENaC is required for the phenotypic switch.

IL-7 Receptor marks entire embryo-derived Mast Cell

Abstract Mast cell (MC) is an unique immune cell displaying wide variety of functions. Recent break through concluded MC generation is mostly provided by embryonic Yolk Sac (YS) and AGM, and post-natal BM HSC does not supply MC (HSC-independent) as our last presentation. Because YS erythro-myeloid progenitor (EMP) provide earliest tissue resident macrophage (brain microglia) and MC together as a common progenitor, and one report shows part of tissue resident macrophages are marked by IL-7 receptor (IL-7R) as an exception of lymphoid essential lineage marking, we hypothesized that MC could be also marked by IL-7R. We tested by utilizing IL-7Rcre/flox-dTom model and found that newborn to young peritoneal cavity (PerC) and skin MCs, surprisingly, exhibited more than 90% dTom positivity (90.8 ± 3.1%). IL-7R protein was not expressed on the mouse PerC, skin MCs and E12.5 Fetal Liver MC progenitors. To confirm functional IL-7R involvement for the MC development, we measured MCs in IL-7Rcre/+(Het) and IL-7Rcre/cre (KO) mice. Despite marked reductions of T/B cell counts, PerC MC count was comparable (WT: 2.4 ± 1.6 vs KO: 4.1 ± 2.3 × 10e4), suggesting IL-7R is temporarily expressed in early EMP stage in an only short period. We also tested MC differentiation from the adult BM dTom-negative Lin−Sca+Kit+ (LSK) cells. MC production was comparable between Het and KO and interestingly enough, both WT/KO LSK derived MCs showed least dTom positivity (3.4± 2.5 vs 4.7 ± 3.1 %) whereas more than 70% of macrophages turned dTom positive, suggesting embryonic and adult MC differentiation used different program respectively. Consistently, %dTom in PerC MC was reduced (79.2 ± 8.1 %) in old mice (> 1year), indicating that post-natal de novo MC production is minimum without IL-7R use. Supported by R01AI121197-01A1 NIAID R01AI147685-01 NIAID

Bacillus anthracis peptidoglycan alters human M2-like macrophage phenotype and efferocytic function in the presence of human serum

Abstract Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen associated molecular pattern contributing to anthrax pathology including coagulopathy in late stage disease. Lymphocyte apoptosis is an established phenomenon in bacterial sepsis, including fulminant anthrax. Clearance of apoptotic cells (efferocytosis) is thought to be mediated by tissue-resident macrophages (MF), modeled in vitro as M2-like MF. Circulating innate cells need serum opsonization of PGN for cellular activation, but opsonization requirements for human MF activation have not been reported. Herein, we tested the hypothesis that PGN requires human serum opsonization for MF activation and inhibits human M2-like MF efferocytosis of apoptotic human neutrophils. MF were differentiated in vitro with M-CSF and polarized to M2 phenotype with dexamethasone. Cytokine and soluble receptor levels were assessed by ELISA and Luminex assays. Cell surface markers and fraction of MF containing fluorescently-labeled apoptotic human neutrophils were assessed by flow cytometry. M2-like MF recognition of PGN was enhanced in the presence of human but not bovine serum, noted by increased secretion of TNF-a and IL-10 in PGN-treated culture supernatants. Efferocytosis, and cell surface expression of MerTK, Tyro3, Axl, αVβ5, CD36 and Tim-3, all pro-efferocytic receptors, was significantly downregulated by pre-treatment of MF with PGN. Increases in soluble pro-efferocytic receptors were detected in PGN-treated MF culture supernatants. We conclude that PGN impairs efferocytosis by human M2-like macrophages, likely by down-regulating the expression of cell surface receptors known to signal apoptotic cell engulfment. Supported by NIH (U19AI062629).

Phenotypic and transcriptomic profiles of tumor-conditioned macrophages in dogs, and the translational value of canine cancer immunology research.

Abstract Tumor-associated macrophages (TAMs) are heterogeneous and abundantly present in the tumor stroma. They derive from tissue-resident macrophages and recruited monocytes and play a central role in several aspects of cancer. In murine models, TAMs promote invasion, intravasation, circulatory survival and extravasation of tumor cells. TAMs also promote tumor progression and angiogenesis, remodel the tumor microenvironment, and modulate the adaptive immune system. Evidence suggests TAMs are vital for establishing the premetastatic niche. Historically, family-owned dogs have proven to be reliable cancer models. In contrast to laboratory animals, dogs develop cancer naturally and commonly, co-inhabit our environment, are genetically outbred and immunologically experienced. We hypothesized that canine tumor conditioned monocyte-derived macrophages (TCMs) share many characteristics with M2 macrophages and have a tumor-promoting phenotype as in humans. We collected blood from healthy dogs, isolated peripheral blood monocytes and differentiated them in vitro using tumor conditioned media from three canine cancer lines and M-CSF. Three TCM populations were generated and compared with M1 and M2 macrophages. We assessed the macrophages using multicolor flow cytometry and RNA-sequencing. All TCM showed an increased expression of two M2 markers (CD209a and FcɛRI), while 2/3 TCM populations had increased expression of two additional M2 markers (CD206 and CD11d). Transcriptomic analysis is ongoing. Results so far support the hypothesis that canine TCMs share many similarities with human TCMs, further warranting the use of dogs as cancer immunology models. Supported by grant from Agria Animal Insurances (N-2020-0061) and The Research fund for cancer in dogs (Oslo, Norway)