BACKGROUND: Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients.
METHODS: Fourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients\' informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFβ1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test.
RESULTS: Cultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells).
CONCLUSIONS: In cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFβ1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

To determine the role that the IL-4/IL13 receptor plays in the development of alternatively activated macrophages (AAM or M2) and their role in the regulation of immunity to the extraintestinal phase of the helminth parasite Taenia crassiceps, we followed the infection in a mouse strain lacking the IL-4Rα gene (IL-4Rα-/-) and in the macrophage/neutrophil-specific IL-4Rα-deficient mouse strain (LysMcreIL-4Rα-/lox or cre/LoxP). While 100% of T. crassiceps-infected IL-4Rα+/+ (WT) mice harbored large parasite loads, more than 50% of th eIL-4Rα-/- mice resolved the infection. Approximately 88% of the LysMcreIL-4Rα-/lox mice displayed a sterilizing immunity to the infection. The remaining few infected cre/LoxP mice displayed the lowest number of larvae in their peritoneal cavity. The inability of the WT mice to control the infection was associated with antigen-specific Th2-type responses with higher levels of IgG1, IL-4, IL-13, and total IgE, reduced NO production, and increased arginase activity. In contrast, IL-4Rα-/- semi-resistant mice showed a Th1/Th2 combined response. Furthermore, macrophages from the WT mice displayed higher transcripts for Arginase-1 and RELM-α, as well as increased expression of PD-L2 with robust suppressive activity over anti-CD3/CD28 stimulated T cells; all of these features are associated with the AAM or M2 macrophage phenotype. In contrast, both the IL-4Rα-/- and LysMcreIL-4Rα-/lox mice did not fully develop AAM or display suppressive activity over CD3/CD28 stimulated T cells, reducing PDL2 expression. Additionally, T-CD8+ but no T-CD4+ cells showed a suppressive phenotype with increased Tim-3 and PD1 expression in WT and IL-4Rα-/-, which were absent in T. crassiceps-infected LysMcreIL-4Rα-/lox mice. These findings demonstrate a critical role for the IL-4 signaling pathway in sustaining AAM and its suppressive activity during cysticercosis, suggesting a pivotal role for AAM in favoring susceptibility to T. crassiceps infection. Thus, the absence of these suppressor cells is one of the leading mechanisms to control experimental cysticercosis successfully.

BACKGROUND: There is an increase in the global incidence of allergies. The hygiene hypothesis and the old friend hypothesis reveal that helminths are associated with the prevalence of allergic diseases. The therapeutic potential of Trichinella spiralis is recognized; however, the stage at which it exerts its immunomodulatory effect is unclear.
METHODS: We evaluated the differentiation of bone marrow-derived macrophages stimulated with T spiralis excretory-secretory products. Based on an ovalbumin-induced murine model, T spiralis was introduced during 3 allergy phases. Cytokine levels and immune cell subsets in the lung, spleen, and peritoneal cavity were assessed.
RESULTS: We found that T spiralis infection reduced lung inflammation, increased anti-inflammatory cytokines, and decreased Th2 cytokines and alarms. Recruitment of eosinophils, CD11b+ dendritic cells, and interstitial macrophages to the lung was significantly suppressed, whereas Treg cells and alternatively activated macrophages increased in T spiralis infection groups vs the ovalbumin group. Notably, when T spiralis was infected prior to ovalbumin challenge, intestinal adults promoted proportions of CD103+ dendritic cells and alveolar macrophages.
CONCLUSIONS: T spiralis strongly suppressed type 2 inflammation, and adults maintained lung immune homeostasis.

Alveolar macrophages (alvMs) play an important role for maintenance of lung function by constant removal of cellular debris in the alveolar space. They further contribute to defense against microbial or viral infections and limit tissue damage during acute lung injury. alvMs arise from embryonic progenitor cells, seed the alveoli before birth, and have life-long self-renewing capacity. However, recruited monocytes may also help to restore the alvM population after depletion caused by toxins or influenza virus infection. At present, the population dynamics and cellular plasticity of alvMs during allergic lung inflammation is poorly defined. To address this point, we used a mouse model of Aspergillus fumigatus-induced allergic lung inflammation and observed that Th2-derived IL-4 and IL-13 caused almost complete disappearance of alvMs. This effect required STAT6 expression in alvMs and also occurred in various other settings of type 2 immunity-mediated lung inflammation or administration of IL-4 complexes to the lung. In addition, Th2 cells promoted conversion of alvMs to alternatively activated macrophages and multinucleated giant cells. Given the well-established role of alvMs for maintenance of lung function, this process may have implications for resolution of inflammation and tissue homeostasis in allergic asthma.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited therapeutic options. Macrophages, particularly alternatively activated macrophages (M2), have been recognized to contribute to the pathogenesis of pulmonary fibrosis. Therefore, targeting macrophages might be a viable therapeutic strategy for IPF. Herein, we report a potential nanomedicine-based gene therapy for IPF by modulating macrophage M2 activation. In this study, we illustrated that the levels of pleckstrin homology and FYVE domain containing 1 (Plekhf1) were increased in the lungs originating from IPF patients and PF mice. Further functionality studies identified the pivotal role of Plekhf1 in macrophage M2 activation. Mechanistically, Plekhf1 was upregulated by IL-4/IL-13 stimulation, after which Plekhf1 enhanced PI3K/Akt signaling to promote the macrophage M2 program and exacerbate pulmonary fibrosis. Therefore, intratracheal administration of Plekhf1 siRNA-loaded liposomes could effectively suppress the expression of Plekhf1 in the lungs and notably protect mice against BLM-induced lung injury and fibrosis, concomitant with a significant reduction in M2 macrophage accumulation in the lungs. In conclusion, Plekhf1 may play a crucial role in the pathogenesis of pulmonary fibrosis, and Plekhf1 siRNA-loaded liposomes might be a promising therapeutic approach against pulmonary fibrosis.

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.

Capillaria hepatica is a seriously neglected zoonotic parasite, which infects the liver of mammalian hosts, causing fibrosis or even hepatic failure. At present, the immune responses elicited by C. hepatica are not fully understood, and the role(s) of the programmed death 1 (PD-1) signaling pathway in the context of C. hepatica-induced pathology are not known. In this study, we identify that the late stage of infection with C. hepatica-especially the egg-derived antigens-modulates the host immune responses to promote alternatively activated macrophage (M2) polarization and programmed death ligand 2 (PD-L2) expression. The PD-L2-expressing alternatively activated M2 macrophages play an important role in maintaining Th2-biased regulatory immune responses, which may facilitate the survival of parasitic worms or eggs within the infected liver and reduce the liver pathology caused by the egg granulomas. Treatment with anti-PD-L2 antibody had no effect on the survival of parasitic eggs but deteriorated the pathology of egg granulomas. The obtained results suggest that PD-1/PD-L2 signaling, which is involved in alternative macrophage polarization, determines the immune response pattern and the immunopathology, consequently determining the outcome of the parasitic infection.

The phenotype of tumor-associated macrophages may be critical for tumor immunity, angiogenesis, and clinical disease outcome. Here, we elucidated the prognostic significance of the neovasculature and macrophages in colorectal cancer. We analyzed the effect of M2 macrophage density on the clinical behavior of 151 primary colorectal carcinomas using CD206 as a marker for type 2 macrophages. Triple immunohistochemical staining (ERG, SMA, and podoplanin) was used for microvessel evaluation. We found that M2 macrophages in colorectal cancer did not have a direct association with metastatic behavior. However, high numbers of CD206 tumor-associated macrophages correlated positively with recurrence-free interval duration (P=0.005). Fewer macrophages in the tumor microenvironment resulted in insufficient coverage of newly formed vessels by pericytes (P=0.011), and a high number of pericyte-impaired microvessels correlated with metastatic behavior (P<0.001). These results suggested that type 2 macrophages had a role in limiting the metastatic process by affecting vascular maturity and normalization. These findings contribute to understanding complex interactions in the tumor microenvironment and the clinical behavior of colorectal cancer.

Schistosomiasis is a neglected tropical disease caused by worms of the genus Schistosoma spp. The progression of disease results in intense tissue fibrosis and high mortality rate. After egg deposition by adult worms, the inflammatory response is characterized by the robust activation of type 2 immunity. Monocytes and macrophages play critical roles during schistosomiasis. Inflammatory Ly6Chigh monocytes are recruited from the blood to the inflammatory foci and differentiate into alternatively activated macrophages (AAMs), which promote tissue repair. The common chain of β2-integrins (CD18) regulates monocytopoiesis and mediates resistance to experimental schistosomiasis. There is still limited knowledge about mechanisms controlled by CD18 that impact monocyte development and effector cells such as macrophages during schistosomiasis. Here, we show that CD18low mice chronically infected with S. mansoni display monocyte progenitors with reduced proliferative capacity, resulting in the accumulation of the progenitor cell denominated proliferating-monocyte (pMo). Consequently, inflammatory Ly6Chigh and patrolling Ly6Clow monocytes are reduced in the bone marrow and blood. Mechanistically, low CD18 expression decreases Irf8 gene expression in pMo progenitor cells, whose encoded transcription factor regulates CSFR1 (CD115) expression on the cell surface. Furthermore, low CD18 expression affects the accumulation of inflammatory Ly6Chigh CD11b+ monocytes in the liver while the adoptive transference of these cells to infected-CD18low mice reduced the inflammatory infiltrate and fibrosis in the liver. Importantly, expression of Il4, Chil3l3 and Arg1 was downregulated, CD206+PD-L2+ AAMs were reduced and there were lower levels of IL-10 in the liver of CD18low mice chronically infected with S. mansoni. Overall, these findings suggest that CD18 controls the IRF8-CD115 axis on pMo progenitor cells, affecting their proliferation and maturation of monocytes. At the same time, CD18 is crucial for the appropriate polarization and function of AAMs and tissue repair during chronic schistosomiasis.

To address the role of methyl-CpG-binding domain 2 (MBD2) in the pathogenesis of asthma and its potential as a target for the asthmatic therapy.
Studies were conducted in asthmatic patients and macrophage-specific Mbd2 knockout mice to dissect the role of MBD2 in asthma pathogenesis. Additionally, RNAi-based therapy with Mbd2 siRNA-loaded liposomes was conducted in an ovalbumin (OVA)-induced allergic airway inflammation mouse model.
Asthmatic patients and mice challenged with OVA exhibited upregulated MBD2 expression in macrophages, especially in alternatively activated (M2) macrophages. In particular, macrophage-specific knockout of Mbd2 protected mice from OVA-induced allergic airway inflammation and suppressed the M2 program. Notably, intratracheal administration of liposomes carrying Mbd2 siRNA decreased the expression of Mbd2 and prevented OVA-induced allergic airway inflammation in mice, as indicated by the attenuated airway inflammation and mucus production.
The above data indicate that Mbd2 implicates in the pathogenesis of asthma predominantly by regulating the polarization of M2 macrophages, which supports that Mbd2 could be a viable target for treatment of asthma in clinical settings.