Visualizing Macrophage Phenotypes and Polarization in Diseases:From Biomarkers to Molecular Probes

Macrophage is a kind of immune cell and performs multiple functions including pathogen phagocytosis,antigen presentation and tissue remodeling.To fulfll their functionally distinct roles,macrophages undergo polarization towards a spectrum of phenotypes,particularly the classically activated(M1)and alternatively activated(M2)subtypes.However,the binary M1/M2 phenotype fails to capture the complexity of macrophages subpopulations in vivo.Hence,it is crucial to employ spatiotemporal imaging techniques to visualize macrophage phenotypes and polarization,enabling the monitoring of disease progression and assessment of therapeutic responses to drug candidates.This review begins by discussing the origin,function and diversity of macrophage under physiological and pathological conditions.Subsequently,we summarize the identifed macrophage phenotypes and their specifc biomarkers.In addition,we present the imaging probes locating the lesions by visualizing macrophages with specifc phenotype in vivo.Finally,we discuss the challenges and prospects associated with monitoring immune microenvironment and disease progression through imaging of macrophage phenotypes.

Interactions between myoblasts and macrophages under high glucose milieus result in inflammatory response and impaired insulin sensitivity

BACKGROUND Skeletal muscle handles about 80% of insulin-stimulated glucose uptake and become the major organ occurring insulin resistance(IR).Many studies have confirmed the interactions between macrophages and skeletal muscle regulated the inflammation and regeneration of skeletal muscle.However,despite of the decades of research,whether macrophages infiltration and polarization in skeletal muscle under high glucose(HG)milieus results in the development of IR is yet to be elucidated.C2C12 myoblasts are well-established and excellent model to study myogenic regulation and its responses to stimulation.Further exploration of macrophages'role in myoblasts IR and the dynamics of their infiltration and polarization is warranted.AIM To evaluate interactions between myoblasts and macrophages under HG,and its effects on inflammation and IR in skeletal muscle.METHODS We detected the polarization status of macrophages infiltrated to skeletal muscles of IR mice by hematoxylin and eosin and immunohistochemical staining.Then,we developed an in vitro co-culture system to study the interactions between myoblasts and macrophages under HG milieus.The effects of myoblasts on macrophages were explored through morphological observation,CCK-8 assay,Flow Cytometry,and enzyme-linked immunosorbent assay.The mediation of macrophages to myogenesis and insulin sensitivity were detected by morphological observation,CCK-8 assay,Immunofluorescence,and 2-NBDG assay.RESULTS The F4/80 and co-localization of F4/80 and CD86 increased,and the myofiber size decreased in IR group(P<0.01,g=6.26).Compared to Mc group,F4/80+CD86+CD206-cells,tumor necrosis factor-α(TNFα),inerleukin-1β(IL-1β)and IL-6 decreased,and IL-10 increased in McM group(P<0.01,g>0.8).In McM+HG group,F4/80+CD86+CD206-cells,monocyte chemoattractant protein 1,TNFα,IL-1βand IL-6 were increased,and F4/80+CD206+CD86-cells and IL-10 were decreased compared with Mc+HG group and McM group(P<0.01,g>0.8).Compered to M group,myotube area,myotube number and E-MHC were increased in MMc group(P<0.01,g>0.8).In MMc+HG group,myotube area,myotube number,E-MHC,GLUT4 and glucose uptake were decreased compared with M+HG group and MMc group(P<0.01,g>0.8).CONCLUSION Interactions between myoblasts and macrophages under HG milieus results in inflammation and IR,which support that the macrophage may serve as a promising therapeutic target for skeletal muscle atrophy and IR.

Light-induced dynamic RGD pattern for sequential modulation of macrophage phenotypes

Due to the critical roles of macrophage in immune response and tissue repair,harnessing macrophage phenotypes dynamically to match the tissue healing process on demand attracted many attentions.Although there have developed many advanced platforms with dynamic features for cell manipulation,few studies have designed a dynamic chemical pattern to sequentially polarize macrophage phenotypes and meet the immune requirements at various tissue repair stages.Here,we propose a novel strategy for spatiotemporal manipulation of macrophage phenotypes by a UV-induced dynamic Arg-Gly-Asp(RGD)pattern.By employing a photo-patterning technique and the specific interaction between cyclodextrin(CD)and azobenzene-RGD(Azo-RGD),we prepared a polyethylene glycol-dithiol/polyethylene glycol-norbornene(PEG-SH/PEG-Nor)hydrogel with dynamic RGD-patterned surface.After irradiation with 365-nm UV light,the homogeneous RGD surface was transformed to the RGD-patterned surface which induced morphological transformation of macrophages from round to elongated and subsequent phenotypic transition from pro-inflammation to anti-inflammation.The mechanism of phenotypic polarization induced by RGD pattern was proved to be related to Rho-associated protein kinase 2(ROCK2).Sequential modulation of macrophage phenotypes by the dynamic RGD-patterned surface provides a remote and non-invasive strategy to manipulate immune reactions and achieve optimized healing outcomes.

Sulforaphane attenuates dextran sodium sulphate induced intestinal inflammation via IL-10/STAT3 signaling mediated macrophage phenotype switching

Innate immunity,particularly macrophages,is critical for intestinal homeostasis.Sulforaphane,a dietary isothiocyanate from cruciferous vegetables,has been reported to protect against intestinal inflammation.However,the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet.In this study,sulforaphane effectively attenuated dextran sodium sulphate(DSS)induced intestinal inflammation in murine model.Of note,sulforaphane skewed the switching from classically(M1)to alternatively(M2)activated phenotype both in intestinal and bone marrow-derived macrophages(BMDMs).The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide(LPS)plus interferon gamma-γ(IFN-γ)were suppressed by sulforaphane while M2 marker gene expression levels were improved.This resulted in alteration of inflammatory mediators,particularly interleukin-10(IL-10),both in colon tissues and culture medium of BMDMs.Subsequently,IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling.This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections.Moreover,anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation.Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.

The miR-21-5p enriched in the apoptotic bodies of M2 macrophage-derived extracellular vesicles alleviates osteoarthritis by changing macrophage phenotype

Macrophages (Mφs) play a crucial role in the pathological progression of osteoarthritis (OA) by regulating inflammation and tissue repair. Decreasing pro-inflammatory M1-Mφs and increasing anti-inflammatory M2-Mφs can alleviate OA-related inflammation and promote cartilage repair. Apoptosis is a natural process associated with tissue repair. A large number of apoptotic bodies (ABs), a type of extracellular vesicle, are produced during apoptosis, and this is associated with a reduction in inflammation. However, the functions of apoptotic bodies remain largely unknown. In this study, we investigated the role of M2-Mφs-derived apoptotic bodies (M2-ABs) in regulating the M1/M2 balance of macrophages in a mouse model of OA. Our data show that M2-ABs can be targeted for uptake by M1-Mφs, and this reprograms M1-to-M2 phenotypes within 24 h. The M2-ABs significantly ameliorated the severity of OA, alleviated the M1-mediated pro-inflammatory environment, and inhibited chondrocyte apoptosis in mice. RNA-seq revealed that M2-ABs were enriched with miR-21–5p, a microRNA that is negatively correlated with articular cartilage degeneration. Inhibiting the function of miR-21–5p in M1-Mφs significantly reduced M2-ABs-guided M1-to-M2 reprogramming following in vitro cell transfection. Together, these results suggest that M2-derived apoptotic bodies can prevent articular cartilage damage and improve gait abnormalities in OA mice by reversing the inflammatory response caused by M1 macrophages. The mechanism underlying these findings may be related to miR-21-5p-regulated inhibition of inflammatory factors. The application of M2-ABs may represent a novel cell therapy, and could provide a valuable strategy for the treatment of OA and/or chronic inflammation.

Polarizing the immune system towards neuroprotection in brain ischemia

The development of ischemic brain damage is dramatically affected by the immune system,whose activation occurs immediately after the insult and may last for several days,involving a complex interplay between soluble and cellular mediators（Amantea et al.,2015）.Accordingly,recent expression profiling studies have revealed that the majority of the genes modulated in the blood of stroke patients participate in the regulation of innate immune responses（Brooks et al.,2014）.

Response of macrophages in rat skeletal muscle after eccentric exercise

Purpose： Macrophages are known to be important for healing numerous injured tissues depending on their functional phenotypes in response to different stimuli. The objective of this study was to reveal macrophage phenotypic changes involved in exercise-induced skeletal muscle injury and regeneration. Methods： Adult male Sprague-Dawley rats experienced one session of downhill running （16~ decline, 16 m/min） for 90 min. After exercise the blood and soleus muscles were collected at 0 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 w and 2 w after exercise, separately. Results： It was showed that CD6B＋ M1 macrophages mainly infiltrated into musc｜e necrotic sites at 1-3 d, while CD163＋ M2 macrophages were present in muscles from 0 h to 2 weeks after exercise. Using transmission electron microscopy, we observed activated satellite cells 1 d after exercise. Thl-associated transcripts of iNOS and Cc12 were inhibited post exercise, while COX-2 mRNA was dramatically increased 12 h after running （p 〈 0.01 ）. M2 phenotype marker Arg-1 increased 12 h and 3 d （p 〈 0.05, p 〈 0.01 ） after exercise, and Clecl0a and Mrc2 were up-regulated in muscles 12 h following exercise （p 〈 0.05, p 〈 0.05）. Conclusion： The data demonstrate the dynamic patterns of macrophage phenotype in skeletal muscle upon eccentric exercise stimuli, and M1 and M2 phenotypes perform different functions during exercise- induced skeletal muscle injury and recovery.

Regulation of the Macrophage Phenotype on Titanium Metal by Surface Modification

The regulation of macrophage phenotype(M1/M2)is very important for tissue repair.The macrophage phenotypes could be affected by the physical and chemical parameters of implant surface.The aim of this study was to investigate the effects of surface modifications of titanium metals on macrophage phenotype.The medical pure titanium metals(PT-Ti)subjected to Anodic Oxidation(AO-Ti),Sand Blasting/acid etching(SLA-Ti)and Plasma-sprayed HA coating(HA coating-Ti)were used for regulating the phenotype of macrophage.The results showed that the Raw264.7 cells of AO-Ti groups had no obvious pseudopodia and could spread evenly in all directions.The levels of IL-1βand TNF-α,which belong to pro-inflammatory genes,expressed by the cells on AO-Ti groups were the lowest among all of the modified titanium groups.But,the levels of IL-10 and TGF-β,which belong to anti-inflammatory genes,expressed on AO-Ti groups were much higher than those on the other groups.Furthermore,the AO-Ti could regulate the expression of SOCS-1 and SOCS-3 to affect the active of NF-κB signaling.The gene expression results of macrophages showed that the AO-Ti was more conductive to inhibit the expression of M1-related genes and promote the expression of M2-related genes in an inflammatory environment.The AO-Ti was more beneficial to tissue repair than other modified titanium metals.The results showed that the anodic oxidation is an effective method to regulate the phenotype of macrophages.

In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection

Respiratory syncytial virus(RSV)is the most common cause of viral bronchiolitis among children worldwide,yet there is no vaccine for RSV disease.This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles(CNP)to modulate reactive oxygen(ROS)and nitrogen(RNS)species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo.Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods,respectively.Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential.In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages.Specifically,cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels.Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFαand IL-12p70,while simultaneously decreasing M2 CD206 expression.Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice.Notably,cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation,avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils,which are associated with RSV-mediated inflammation.In conclusion,we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.