Innate type 2 lymphocytes trigger an inflammatory switch in alveolar macrophages.

Macrophage fusion and multinucleated giant cell formation, surface morphology

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.

IRF4 exacerbates pulmonary inflammation in bronchopulmonary dysplasia mice model by regulating macrophage polarization and phagocytosis.

AIMTo investigate the role of interferon regulatory factor 5 (IRF5) in reversing polarization of lung macrophages during severe acute pancreatitis (SAP) in vitro.METHODSA mouse SAP model was established by intraperitoneal (ip) injections of 20 μg/kg body weight caerulein. Pathological changes in the lung were observed by hematoxylin and eosin staining. Lung macrophages were isolated from bronchoalveolar lavage fluid. The quantity and purity of lung macrophages were detected by fluorescence-activated cell sorting and evaluated by real-time polymerase chain reaction (RT-PCR). They were treated with IL-4/IRF5 specific siRNA (IRF5 siRNA) to reverse their polarization and were evaluated by detecting markers expression of M1/M2 using RT-PCR.RESULTSSAP associated acute lung injury (ALI) was induced successfully by ip injections of caerulein, which was confirmed by histopathology. Lung macrophages expressed high levels of IRF5 as M1 phenotype during the early acute pancreatitis stages. Reduction of IRF5 expression by IRF5 siRNA reversed the action of macrophages from M1 to M2 phenotype in vitro. The expressions of M1 markers, including IRF5 (S + IRF5 siRNA vs S + PBS, 0.013 ± 0.01 vs 0.054 ± 0.047, P < 0.01), TNF-α (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.019 ± 0.018, P < 0.001), iNOS (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.026 ± 0.018, P < 0.001) and IL-12 (S + IRF5 siRNA vs S + PBS, 0.000005 ± 0.00004 vs 0.024 ± 0.016, P < 0.001), were decreased. In contrast, the expressions of M2 markers, including IL-10 (S + IRF5 siRNA vs S + PBS, 0.060 ± 0.055 vs 0.0230 ± 0.018, P < 0.01) and Arg-1 (S + IRF5 siRNA vs S + PBS, 0.910 ± 0.788 vs 0.0036 ± 0.0025, P < 0.001), were increased. IRF5 siRNA could reverse the lung macrophage polarization more effectively than IL-4.CONCLUSIONTreatment with IRF5 siRNA can reverse the pancreatitis-induced activation of lung macrophages from M1 phenotype to M2 phenotype in SAP associated with ALI.

RationaleSeasonal influenza A virus infection causes at least 20,000–50,000 deaths in the United States each year and causes disproportionate morbidity and mortality in older individuals. We and others have found similarly disproportionate mortality in aged mice infected with influenza A. An important role for alveolar macrophages in the response to influenza A infection is recognized. Strategies to therapeutically target alveolar macrophages during lung injury have considered that they are a single population of monocyte‐derived cells. This paradigm has been challenged by studies that identified tissue‐resident alveolar macrophages (TR‐AM) as highly specialized cells that populate the lung shortly after birth and persist over the lifespan.MethodsWe aged bone marrow chimeric C57Bl/6 (CD45.1 donor into CD45.2 host) mice generated with thoracic shielding followed by busulfan depletion of the remaining bone marrow. The resulting chimeric mice (&gt;99% donor monocytes in blood, &gt;99 % recipient in tissue resident alveolar macrophages), were treated with influenza A (A/WSN/33) virus, or bleomycin.ResultsSevere injury early in life alters the alveolar macrophage landscape during aging. We treated shielded‐bone‐marrow chimeric mice with influenza A or bleomycin and measured the ratio between Mo‐AM and TR‐AM 10 months later. We found that TR‐AM were remarkably stable, constituting &gt;95% of alveolar macrophages in naïve mice at 14 months of age. In contrast, in mice infected with influenza A (A/WSN/33) virus, 100pfu at 8 weeks post bone marrow transfer (4 months of age), approximately 50% of the alveolar macrophages were monocyte‐derived 10 months later. To determine whether this finding was unique to influenza A, we treated a separate cohort of shielded‐bone‐marrow chimeric mice with bleomycin and obtained similar results. Next, we sought to determine whether the differences in gene expression between TR‐AM and Mo‐AM we observed during injury and fibrosis persisted over the lifespan. Ten months after bleomycin treatment or influenza A infection, TR‐AM and Mo‐AM were not disgtinguishable by flow cytometry. A comparison of the transcriptomes of TR‐AM and Mo‐AM 10 months after bleomycin administration revealed only 101 differentially expressed genes.ConclusionThis finding suggests that a severe injury early in life can permanently reshape the alveolar macrophage landscape with respect to its developmental origins. If monocyte‐derived alveolar macrophages differ in their response to challenge, this might explain some of the age‐related susceptibility to influenza A infection.Support or Funding InformationAG049665, HL071643, ES013995, The Veterans Administration, DOD W81XWH‐15‐1‐0215

While antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) and has enabled people living with HIV (PLWH) to achieve near-normal life expectancies, an HIV cure remains elusive due to the presence of HIV reservoirs. Furthermore, compared with individuals in the general population, PLWH support a higher burden of multimorbidity, including pulmonary diseases of both an infectious and non-infection nature, which may be a consequence of the formation of HIV reservoirs. Their gut, lymph nodes, brain, testes and lungs constitute important anatomic sites for the reservoirs. While CD4+ T-cells, and particularly memory CD4+ T-cells, are the best characterized cellular HIV reservoirs, tissue resident macrophages (TRM) and alveolar macrophages (AM) also harbor HIV infection. AM are the most abundant cells in bronchoalveolar (BAL) fluid in healthy conditions, and act as sentinels in the alveolar space by patrolling and clearing debris, microbes and surfactant recycling. Long-lived tissue-resident AM of embryonic origin have the capacity of self-renewal without replenishment from peripheral monocytes. As in other tissues, close cell-cell contacts in lungs also provide a milieu conducive for cell-to-cell spread of HIV infection and establishment of reservoirs. As lungs are in constant exposure to antigens from the external environment, this situation contributes to pro-inflammatory phenotype rendering pulmonary immune cells exhausted and senescent-an environment facilitating HIV persistence. Factors such as tobacco and e-cigarette smoking, lung microbiome dysbiosis and respiratory co-infections further drive antigenic stimulation and HIV replication. HIV replication, in turn, contributes to ongoing inflammation and clonal expansion. Herein, the potential role of AM in HIV persistence is discussed. Furthermore, their contribution towards pulmonary inflammation and immune dysregulation, which may in turn render PLWH susceptible to chronic lung disease, despite ART, is explored. Finally, strategies to eliminate HIV-infected AM are discussed.

While antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) and has enabled people living with HIV (PLWH) to achieve near-normal life expectancies, an HIV cure remains elusive due to the presence of HIV reservoirs. Furthermore, compared with individuals in the general population, PLWH support a higher burden of multimorbidity, including pulmonary diseases of both an infectious and non-infection nature, which may be a consequence of the formation of HIV reservoirs. Their gut, lymph nodes, brain, testes and lungs constitute important anatomic sites for the reservoirs. While CD4+ T cells, and particularly memory CD4+ T cells, are the best characterized cellular HIV reservoirs, tissue resident macrophages (TRM) and alveolar macrophages (AM) also harbor HIV infection. AM are the most abundant cells in bronchoalveolar (BAL) fluid in healthy conditions, and act as sentinels in the alveolar space by patrolling and clearing debris, microbes and surfactant recycling. Long-lived tissue-resident AM of embryonic origin have the capacity of self-renewal without replenishment from peripheral monocytes. As in other tissues, close cell-cell contacts in lungs also provide a milieu conducive for cell-to-cell spread of HIV infection and establishment of reservoirs. As lungs are in constant exposure to antigens from the external environment, this situation contributes to pro-inflammatory phenotype rendering pulmonary immune cells exhausted and senescent-an environment facilitating HIV persistence. Factors such as tobacco and e-cigarette smoking, lung microbiome dysbiosis and respiratory coinfections further drive antigenic stimulation and HIV replication. HIV replication, in turn, contributes to ongoing inflammation and clonal expansion. Herein, the potential role of AM in HIV persistence is discussed. Furthermore, their contribution towards pulmonary inflammation and immune dysregulation, which may in turn render PLWH susceptible to chronic lung disease, despite ART, is explored. Finally, strategies to eliminate HIV-infected AM are discussed.

Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

Intracellular bacteria, such as the US biological Class B bacterium Coxiella burnetii, are known to infect alveolar macrophages (AM). However, it is not known if a particular subset of AM, within a relatively heterogeneous population of lung macrophages, is particularly susceptible to bacterial infection. Nor is it known whether certain AM are less efficient at resolving infection. We hypothesized that, as a result of tightly regulated immune function, tissue resident AM would prove susceptible to C. burnetii infection and provide a required niche for bacterial persistence. Dye labeling techniques allowed discrimination of AM subsets during the course of infection. Infected cell populations were identified immunohistochemically and through the use of fluorescent protein expressing C. burnetii. We report here that the tissue resident AM are the predominantly infected AM subset, even at low infective doses; these cells remain infected at least nine days after the onset of infection, indicating a permissive niche for bacterial infection. These tissue resident AM undergo a distinguishing phenotypic change during the progression of infection and account for the cell-type predominating in the early uptake of Coxiella. Identification of the AM subset, both susceptible to and permissive of Coxiella burnetii infection, marks an important step in understanding Coxiella pathogenesis providing insight into general pulmonary innate immune function in response to intracellular bacteria.

Inhibition of microglia multinucleated giant cell formation and induction of differentiation by GM-CSF using a porcine in vitro model

IntroductionIdiopathic pulmonary fibrosis is a progressive lung disease with a poor prognosis. Alveolar macrophages (AMs) are essential for maintaining lung homeostasis and play a significant role in the development of lung fibrosis. Tissue-Resident Alveolar Macrophages (TR-AMs), which originate from embryonic progenitors, can self-renew locally in a steady state, independent of hematopoiesis. During fibrogenesis, circulating monocytes rapidly migrate into the lungs and differentiate into monocyte-derived AMs (Mo-AMs). MicroRNAs (miRNAs), small non-coding RNAs, are critical for regulating gene expression. Our recent study found that the loss of miRNAs in embryonic progenitors significantly decreased the number of TR-AMs in late-stage embryos, indicating that miRNAs are necessary for TR-AM development. However, the role of miRNAs in the postnatal maintenance of TR-AMs and Mo-AMs, as well as their function in pulmonary fibrosis, remains unclear.Methods and ResultsHere, we demonstrate that deleting miRNAs after birth severely disrupts TR-AM homeostasis and Mo-AM repopulation from the bone marrow following irradiation. The deficiency of miRNAs in TR-AMs and Mo-AMs was linked to diminished bleomycin-induced experimental lung fibrosis. Mechanistically, the absence of miRNAs increased TR-AM apoptosis under both normal and fibrotic conditions. RNA sequencing (RNA-seq) analysis revealed distinct transcriptomic and pathway changes in miRNA-deficient AM subgroups after lung injury. The integration of RNA-seq and miRNA array analyses identified miRNA-mRNA networks in TR-AMs and Mo-AMs in response to bleomycin injury. Ingenuity Pathway Analysis further predicted let-7a, miR-155, and miR-125 as unique upstream regulators of Mo-AM responses to lung fibrosis.ConclusionsOur findings suggest that miRNAs are key epigenetic mediators that differentially regulate the maintenance and function of TR-AMs and Mo-AMs in the pathogenesis of pulmonary fibrosis.

Multinucleated giant cells (MGC) are a prominent feature of some chronic inflammatory states in the lung. These cells are formed by macrophage fusion, but how this process relates to the kinetics of alveolar macrophage (AM) production and proliferation is not clear. In this serial study, we compare AM kinetics and MGC formation after instilling carbon, silica, asbestos, bleomycin, and saline into the lungs of mice. Animals were killed up to 16 weeks later with [3H]thymidine injected 1 h before death. Counts of AM and MGC were carried out after bronchoalveolar lavage, and cell labeling was assessed by autoradiography. All test substances induced an inflammatory response with equal AM numbers recovered up to 2 weeks. Subsequently, the number returned to normal after carbon but remained elevated in the other groups. After carbon the lung structure was normal, there was no increase in AM label, and no MGC formed. Bleomycin-injected lungs progressed to fibrosis with only a brief, small increase in AM labeling and no MGC formation. After silica, and particularly asbestos, the lungs showed fibrosis, and many granulomas with large MGC were seen. Lavaged AM from these lungs showed a significant increase in DNA synthesis after 2 weeks, followed by higher numbers of MGC, none of which were labeled. Labeled AM tended to be free of particles, whereas MGC after 4 weeks contained many particles. The results indicate a relationship between AM proliferation and fusion, whereby AM growth appears to be prerequisite for cell infusion and MGC formation as a feature of granulomatous disease.

BackgroundMonocyte-derived alveolar macrophages (Mo_AMs) are increasingly recognised as potential pathogenic factors for idiopathic pulmonary fibrosis (IPF). While scRNAseq analysis has proven valuable in the transcriptome profiling of Mo_AMs, the integration analysis of multi-omics may provide additional dimensions of understanding of these cellular populations.MethodsWe performed multi-omics analysis on 116 scRNAseq, 119 bulkseq and five scATACseq lung tissue samples from IPF. We built a large-scale IPF scRNAseq atlas and conducted the Monocle 2/3 as well as the Cellchat to explore the developmental path and intercellular communication on Mo_AMs. We also reported the difference in metabolisms, tissue repair and phagocytosis between Mo_AMs and tissue-resident alveolar macrophages (TRMs). To determine whether Mo_AMs affected pulmonary function, we projected clinical phenotypes (FVC%pred) from the bulkseq dataset onto the scRNAseq atlas. Finally, we used scATATCseq to uncover the upstream regulatory mechanisms and determine key drivers in Mo_AMs.ResultsWe identified three Mo_AMs clusters and the trajectory analysis further validated the origin of these clusters. Moreover, via the Cellchat analysis, the CXCL12/CXCR4 axis was found to be involved in the molecular basis of reciprocal interactions between Mo_AMs and fibroblasts through the activation of the ERK pathway in Mo_AMs. SPP1_RecMacs (RecMacs, recruited macrophages) were higher in the low-FVC group than in the high-FVC group. Specifically, compared with TRMs, the functions of lipid and energetic metabolism as well as tissue repair were higher in Mo_AMs than TRMs. But, TRMs may have higher level of phagocytosis than TRMs. SPIB (PU.1), JUNB, JUND, BACH2, FOSL2, and SMARCC1 showed stronger association with open chromatin of Mo_AMs than TRMs. Significant upregulated expression and deep chromatin accessibility of APOE were observed in both SPP1_RecMacs and TRMs.ConclusionThrough trajectory analysis, it was confirmed that SPP1_RecMacs derived from Monocytes. Besides, Mo_AMs may influence FVC% pred and aggravate pulmonary fibrosis through the communication with fibroblasts. Furthermore, distinctive transcriptional regulators between Mo_AMs and TRMs implied that they may depend on different upstream regulatory mechanisms. Overall, this work provides a global overview of how Mo_AMs govern IPF and also helps determine better approaches and intervention therapies.

Rabbit alveolar macrophages (AM) induced by complete Freund's adjuvant have been used as a model for activated AM. We studied the effects of in vivo and in vitro corticosteroid on collagenase release and spontaneous formation of multinucleated giant cells (MGC) in culture with this system. Dexamethasone 10- through 10(-7) molar concentration in vitro inhibited both collagenase release and spontaneous AM fusion. Daily in vivo administration of dexamethasone to as much as 0.128 mg/kg similarly suppressed these AM functions in culture. These studies show that in vitro and in vivo corticosteroids inhibit collagenase release and MGC formation of rabbit AM in culture in doses comparable to those used therapeutically in humans. This model may be useful in examining the mechanisms of cell fusion and functions of MGC in granulomatous lung disease.

Anti-RMA is a murine anti-rat monoclonal antibody that binds to a 120-kD surface membrane antigen expressed primarily by alveolar macrophages. Saline-lavaged alveolar macrophages (AM) formed clusters after incubation with anti-RMA. Anti-RMA produced multinucleated giant cells (MGC) in approximately 15% of adherent AM, and the F (ab')2 fragment of anti-RMA yielded MGC in approximately 9% of AM. The Fab fragment of anti-RMA did not promote MGC formation, nor did the murine anti-rat monoclonal antibodies OX41 and W3/25 (anti-CD4). Although anti-RMA produced a tenfold increase in [3H]thymidine incorporation by AM, it yielded a minimal increase in the number of AM. Autoradiography of AM stimulated with anti-RMA showed heterogeneous labeling of nuclei in MGC, suggesting that 3H-labeled AM may fuse with AM that are not actively synthesizing DNA. These findings suggest that binding of anti-RMA to AM may activate DNA synthesis, and promote clustering and fusion of AM, leading to MGC formation.