M1 Response Research Articles

The contribution of tumor-associated macrophages in glioma neo-angiogenesis and implications for anti-angiogenic strategies.

"Tumor-associated macrophages" (TAMs) form a significant cell population in malignant tumors and contribute to tumor growth, metastasis, and neovascularization. Gliomas are characterized by extensive neo-angiogenesis, and knowledge of the role of TAMs in neovascularization is important for future anti-angiogenic therapies. The phenotypes and functions of TAMs are heterogeneous and more complex than a classification into M1 and M2 inflammation response types would suggest. In this review, we provide an update on the current knowledge of the ontogeny of TAMs, focusing on diffuse gliomas. The role of TAMs in the regulation of the different processes in tumor angiogenesis is highlighted and the most recently discovered mechanisms by which TAMs mediate resistance against current antivascular therapies are mentioned. Novel compounds tested in clinical trials are discussed and brought in relation to different TAM-related angiogenesis pathways. In addition, potential therapeutic targets used to intervene in TAM-regulated tumor angiogenesis are summarized.

IL-10 expression in macrophages from neonates born from obese mothers is suppressed by IL-4 and LPS/INFγ.

Ob-monocytes had decreased levels of mRNA for pro-inflammatory cytokines IL-1β, IL-10, and IL-12B compared with controls. Conversely, Ob-macrophages showed increased levels of mRNA for TNFα, IL-4R, and IL-10 compared with controls. M1 response was comparable between both groups, characterized by an important induction of TNFα and IL-1β. In response to an M2 stimulus, control macrophages showed a decreased expression of inflammatory mediators while Ob-macrophages had an additional suppression of the anti-inflammatory mediator IL-10. Changes in IL-1β (monocytes) and IL-10 (macrophages) in Ob-monocytes were paralleled by changes in their promoter DNA methylation in fetal monocytes. These results suggest that monocyte-derived macrophages from obese newborns show a basal anti-inflammatory phenotype with an unbalanced response to M1 and M2 polarization stimuli. The presence of changes in DNA methylation of key inflammatory genes in neonatal monocytes suggests an intrauterine programing of immune function by maternal obesity.

Increased Activation of M2 Macrophages in 1 Year‐Old Rat Lungs Following Neonatal Hyperoxia Exposure

RationaleMacrophages are activated in response to insult. M1 macrophage polarization is associated with inflammation and tissue deterioration, whereas the M2 macrophage corresponds to wound repair and angiogenesis and has an anti‐inflammatory phenotype. Lipopolysaccharide (LPS) or interferon gamma (IFNγ) promote M1 differentiation and up‐regulate pro‐inflammatory cytokines like interleukin‐ (IL‐) 1β, IL‐6, and IL‐12 whereas M2 differentiation is regulated by IL‐4. Since hyperoxia exposure induces inflammation leading to the pathology seen in bronchopulmonary dysplasia (BPD) through induction of M1 macrophages, we tested the macrophage population in lung tissue of 1 year‐old rats. We postulated an increased activation of M2 macrophages in lungs of hyperoxia‐exposed rats, suggesting a continued wound healing response. Collagen levels were also assessed. We expected to see an increase in collagen levels of hyperoxic rats, suggesting that there is persistent phenotype after one year.MethodsNewborn rats were exposed to 85% hyperoxia (Hx) or normoxia (Nx) (21%) for 14 days and then kept at Nx conditions. At one year, the lung was lavaged with PBS, the left lung was inflated and fixed (4% formaldehyde). Cells were isolated using a 100 μm cell strainer, red blood cells were lysed (ammonium chloride), and the cells frozen in freezing medium. For flow cytometry, the cells were stained for CD45, CD24, CD163, CD80, CD86, CD204, and CD206. Analysis was done with an LSR‐2. Collagen levels were assessed using Masson's trichome‐stained lung sections. A blinded third party divded each image into grids and ranked each square on a scale of 0–3 (0=none, 3=severe) for fibrosis.ResultsAnalysis of CD80 and CD86 showed an increased proportion of the M1 macrophages in the Hx lung compared to the Nx lung (11.7% higher in Hx). The Hx M1 showed more of activation marker CD163 compared to the Nx M1 in the CD24− cell population (12% higher in Hx). Similarly, the proportion of CD204+ M2 was increased within the CD45+ cell population of Hx (6.3% higher in Hx). Comparing M1 to M2 macrophages, Hx exposure shifted the ratio of macrophages toward an M2 response. These findings were supported by the histologic assessment of collagen showing increased lung collagen in Hx compared to Nx rats (p=0.008).ConclusionOne year after neonatal Hx exposure, adult rat lungs demonstrate a macrophage population shift towards M2, suggesting a continued anti‐inflammatory response. Further, the increased level of collagen in Hx rats suggests that there is persistent damage after one year. The data indicates that humans born preterm with BPD may have long term alterations in inflammatory and repair pathways.Support or Funding InformationNHLBI, R01 HL115061‐03, Suppl (Eldridge) & NHLBI, R01 HL115061 (Eldridge)

Modulation of microglia in the retina: new insights into diabetic retinopathy.

During last decades, the diagnosis of diabetes has been associated with several chronic complications such as diabetic retinopathy (DR). Recent studies of DR have revealed an inflammatory component, which precedes the detection of alterations in the visual function. During DR, the inflammatory process presents two opposite roles depending on the polarization of resident immune cells of the retina triggering proinflammatory (M1) or antiinflammatory (M2) actions. In an early stage of DR, the M2 response concurs with the M1 and is able to ameliorate inflammation and delay the progression of the disease. However, during the progression of DR, the M1 response is maintained whereas the M2 declines and, in this scenario, the classical proinflammatory signaling pathways are chronically activated leading to retinal neurodegeneration and the loss of visual function. The M1/M2 responses are closely related to the activation and polarization of microglial cells. This review aims to offer an overview of the recent insights into the role of microglial cells during inflammation in DR. We have focused on the possibility of modulating microglia polarization as a new therapeutic strategy in DR treatments.

Impairment of systemic DHA synthesis affects macrophage plasticity and polarization: implications for DHA supplementation during inflammation

Docosahexaenoic acid (DHA) is an omega-3 fatty acid obtained from the diet or synthesized from alpha-linolenic acid through the action of fatty acid elongases (ELOVL) and desaturases. DHA plays important roles in the central nervous system as well as in peripheral organs and is the precursor of several molecules that regulate resolution of inflammation. In the present study, we questioned whether impaired synthesis of DHA affected macrophage plasticity and polarization both in vitro and in vivo models. For this we investigated the activation status and inflammatory response of bone marrow-derived M1 and M2 macrophages obtained from mice deficient of Elovl2 (Elovl2−/−), a key enzyme for DHA synthesis in mammals. Although both wild type and Elovl2−/− mice were able to generate efficient M1 and M2 macrophages, M1 cells derived from Elovl2−/− mice showed an increased expression of key markers (iNOS, CD86 and MARCO) and cytokines (IL-6, IL-12 and IL-23). However, M2 macrophages exhibited upregulated M1-like markers like CD80, CD86 and IL-6, concomitantly with a downregulation of their signature marker CD206. These effects were counteracted in cells obtained from DHA-supplemented animals. Finally, white adipose tissue of Elovl2−/− mice presented an M1-like pro-inflammatory phenotype. Hence, impairment of systemic DHA synthesis delineates an alteration of M1/M2 macrophages both in vitro and in vivo, with M1 being hyperactive and more pro-inflammatory while M2 less protective, supporting the view that DHA has a key role in controlling the balance between pro- and anti-inflammatory processes.

Differential Macrophage Polarization from Pneumocystis in Immunocompetent and Immunosuppressed Hosts: Potential Adjunctive Therapy during Pneumonia.

We explored differential polarization of macrophages during infection using a rat model of Pneumocystis pneumonia. We observed enhanced pulmonary M1 macrophage polarization in immunosuppressed (IS) hosts, but an M2 predominant response in immunocompetent (IC) hosts following Pneumocystis carinii challenge. Increased inflammation and inducible nitric oxide synthase (iNOS) levels characterized the M1 response. However, macrophage ability to produce nitric oxide was defective. In contrast, the lungs of IC animals revealed a prominent M2 gene signature, and these macrophages effectively elicited an oxidative burst associated with clearance of Pneumocystis In addition, during P. carinii infection the expression of Dectin-1, a critical receptor for recognition and clearance of P. carinii, was upregulated in macrophages of IC animals but suppressed in IS animals. In the absence of an appropriate cytokine milieu for M2 differentiation, Pneumocystis induced an M1 response both in vitro and in vivo The M1 response induced by P. carinii was plastic in nature and reversible with appropriate cytokine stimuli. Finally, we tested whether macrophage polarization can be modulated in vivo and used to help manage the pathogenesis of Pneumocystis pneumonia by adoptive transfer. Treatment with both M1 and M2 cells significantly improved survival of P. carinii-infected IS hosts. However, M2 treatment provided the best outcomes with efficient clearance of P. carinii and reduced inflammation.

Diverse Effects on M1 Signaling and Adverse Effect Liability within a Series of M1 Ago-PAMs.

Both historical clinical and recent preclinical data suggest that the M1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M1 activation. Efforts then shifted to focus on selective activation of M1 via either allosteric agonists or positive allosteric modulators (PAMs). While M1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M1 ago-PAMs. Our data demonstrate that closely related M1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M1 PAMs.

Spinal cord direct current stimulation differentially modulates neuronal activity in the dorsal and ventral spinal cord

Spinal cord direct current stimulation (sDCS) has the potential for promoting motor function after injury through its modulatory actions on sensory processing, reflex functions, the motor cortex (M1) motor map, and motor output. Here we addressed systems-level mechanisms underlying sDCS neuromodulation of spinal circuits activated by M1 and peripheral forelimb electrical stimulation in anesthetized healthy rats. We determined the effects of cathodal and anodal sDCS (c- and a-sDCS) on local field potentials (LFP) and single-unit activity recorded at 32 sites simultaneously within the sixth cervical segment using a silicon multielectrode array. M1 stimulation produced distinctive dorsomedial and ventral LFP responses that showed polarity-dependent sDCS modulation. c-sDCS enhanced and a-sDCS depressed significantly ventral M1 responses; neither modulated dorsal responses significantly. Using evoked changes in β- and γ-oscillations to assay network function, c-sDCS enhanced and a-sDCS reduced oscillation power ventrally. c-sDCS increased and a-sDCS decreased background firing and firing synchrony of recorded pairs of single units. Peripheral stimulation produced a region-dependent response that showed polarity-dependent sDCS modulation. The dorsomedial LFP was unaffected by c-sDCS and weakly suppressed with a-sDCS. Peripheral-evoked unit responses showed limited polarity dependence. Our findings stress that ventral motor network behavior is enhanced by the neuromodulatory actions of c-sDCS. The combined actions of c-sDCS on M1-evoked neural responses and network behavior in the cervical spinal cord help explain the reported enhanced motor effects of this neuromodulation approach and inform the mechanisms of sDCS for promoting motor rehabilitation after spinal cord or brain injury.NEW & NOTEWORTHY Spinal cord direct current stimulation (sDCS) modulates spinal functions and shows potential for neural rehabilitation after motor systems injury. Using a multichannel electrode array, we found that cathodal DCS enhanced, and anodal depressed, M1-evoked local field potentials, network oscillations, neuronal activity, and neuronal synchrony, especially in the ventral horn. With this new understanding, it is hoped that sDCS can be developed into a tunable spinal neuromodulatory tool for promoting function after brain or spinal injury.

Defective interhemispheric inhibition in drug-treated focal epilepsies

BackgroundFocal epilepsies (FEs) arise from a lateralized network, while in generalized epilepsies (GEs) there is a bilateral involvement from the outset. Intuitively, the corpus callosum is the anatomical substrate for interhemispheric spread. ObjectiveWe used transcranial magnetic stimulation (TMS) to explore whether there are any physiological differences in the corpus callosum of drug-treated patients with FE and those with genetic GE (GGE), compared to healthy subjects (HS). MethodsTMS was used to measure the interhemispheric inhibition (IHI) from right-to-left primary motor cortex (M1) and viceversa in 16 patients with FE, 17 patients with GGE and 17 HS. A conditioning stimulus (CS) was given to one M1 10 and 50 ms before a test stimulus delivered to the contralateral M1. Motor evoked potentials (MEPs) were analysed both as a function of the side of stimulation and of the epileptic focus (left-right). ResultsIn HS, IHI was reproducible with suppression of MEPs at ISIs of 10 and 50 ms. Similar effects occurred in GGE patients. FE patients behaved differently, since IHI was significantly reduced bilaterally. When FE patients were stratified according to the side of their epileptic focus, the long-ISI IHI (=50 ms) appeared to be defective only when the CS was applied over the “focal” hemisphere. ConclusionsFE patients had a defective inhibitory response of contralateral M1 to inputs travelling from the “focal” hemisphere that was residual to the drug action. Whilst IHI changes would not be crucial for the GGE pathophysiology, they may represent one key factor for the contralateral spread of focal discharges, and seizure generalization.

Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro

Macrophages play an important role in inflammatory responses; however, miRNA-mediated repolarization of macrophages is essential for fulfilling this function. To clarify a series of changes at the RNA level in alveolar macrophages under normal and inflammatory conditions, bronchial alveolar lavage liquid (BALF) was collected from healthy volunteers or patients with pneumonia. This approach, which differs from that used in previously, provides more accurate information about the states of macrophages in different lung microenvironments. In this study, the density plots of macrophage subtypes (M1 and M2) in the BALF of healthy volunteers differed from that of the patients with pneumonia. The M2 subtype dominated in healthy volunteers and was rapidly repolarized to M1 in response to miRNA-mediated gene regulation. Differential miRNA expression in the two macrophage subtypes revealed lower expression of miR-155 and MIR-146a in patients with pneumonia compared with healthy volunteers; this may be related to inflammation and the use of anti-inflammatory drugs. We also found increased TNF-α and IL-6 expression at the RNA level, while macrophage galactose-type C-type lectin 1 (MGL-1) expression decreased with downregulation of miR-155 and miR-146a expression. These results indicate that the gene regulation mediated by miR-155 and miR-146a contributes to human alveolar macrophage phenotype repolarization, thus leading to an early switch from pro-inflammatory to anti-inflammatory cytokine production.

A Porcine Urinary Bladder Matrix Does Not Recapitulate the Spatiotemporal Macrophage Response of Muscle Regeneration after Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) results in irrecoverable loss of muscle tissue making its repair challenging. VML repair with acellular extracellular matrix (ECM) scaffolds devoid of exogenous cells has shown improved muscle function, but limited de novo muscle fiber regeneration. On the other hand, studies using minced autologous and free autologous muscle grafts have reported appreciable muscle regeneration. This raises the fundamental question whether an acellular ECM scaffold can orchestrate the spatiotemporal cellular events necessary for appreciable muscle fiber regeneration. This study compares the macrophage and angiogenic responses including the remodeling outcomes of a commercially available porcine urinary bladder matrix, MatriStem™, and autologous muscle grafts. The early heightened and protracted M1 response of the scaffold indicates that the scaffold does not recapitulate the spatiotemporal macrophage response of the autograft tissue. Additionally, the scaffold only supports limited de novo muscle fiber formation and regressing vessel density. Furthermore, scaffold remodeling is accompanied by increased presence of transforming growth factor and α-smooth muscle actin, which is consistent with remodeling of the scaffold into a fibrotic scar-like tissue. The limited muscle formation and scaffold-mediated fibrosis noted in this study corroborates the findings of recent studies that investigated acellular ECM scaffolds (devoid of myogenic cells) for VML repair. Taken together, acellular ECM scaffolds when used for VML repair will likely remodel into a fibrotic scar-like tissue and support limited de novo muscle fiber regeneration primarily in the proximity of the injured musculature. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel

Perturbation Predictability Can Influence the Long-Latency Stretch Response.

Perturbations applied to the upper limbs elicit short (M1: 25–50 ms) and long-latency (M2: 50–100 ms) responses in the stretched muscle. M1 is produced by a spinal reflex loop, and M2 receives contribution from multiple spinal and supra-spinal pathways. While M1 is relatively immutable to voluntary intention, the remarkable feature of M2 is that its size can change based on intention or goal of the participant (e.g., increasing when resisting the perturbation and decreasing when asked to let-go or relax following the perturbation). While many studies have examined modulation of M2 between passive and various active conditions, through the use of constant foreperiods (interval between warning signal and a perturbation), it has also been shown that the magnitude of the M2 response in a passive condition can change based on factors such as habituation and anticipation of perturbation delivery. To prevent anticipation of a perturbation, most studies have used variable foreperiods; however, the range of possible foreperiod duration differs between experiments. The present study examined the influence of different variable foreperiods on modulation of the M2 response. Fifteen participants performed active and passive responses to a perturbation that stretched wrist flexors. Each block of trials had either a short (2.5–3.5 seconds; high predictability) or long (2.5–10.5 seconds; low predictability) variable foreperiod. As expected, no differences were found between any conditions for M1, while M2 was larger in the active rather than passive conditions. Interestingly, within the two passive conditions, the long variable foreperiods resulted in greater activity at the end of the M2 response than the trials with short foreperiods. These results suggest that perturbation predictability, even when using a variable foreperiod, can influence circuitry contributing to the long-latency stretch response.

Mechanisms underlying heterologous skin scaffold-mediated tissue remodeling.

Biocompatibility of two newly developed porcine skin scaffolds was assessed after 3, 14, 21 and 90 days of implantation in rats. Both scaffolds showed absence of cells, preservation of ECM and mechanical properties comparable to non-decellularised skin before implantation. Host cell infiltration was much prominent on both scaffolds when compared to Permacol (surgical control). At day 3, the grafts were surrounded by polymorphonuclear cells, which were replaced by a notable number of IL-6-positive cells at day 14. Simultaneously, the number of pro-inflammatory M1-macrophage was enhanced. Interestingly, a predominant pro-remodeling M2 response, with newly formed vessels, myofibroblasts activation and a shift on the type of collagen expression was sequentially delayed (around 21 days). The gene expression of some trophic factors involved in tissue remodeling was congruent with the cellular events. Our findings suggested that the responsiveness of macrophages after non-crosslinked skin scaffolds implantation seemed to intimately affect various cell responses and molecular events; and this range of mutually reinforcing actions was predictive of a positive tissue remodeling that was essential for the long-standing success of the implants. Furthermore, our study indicates that non-crosslinked biologic scaffold implantation is biocompatible to the host tissue and somehow underlying molecular events involved in tissue repair.

An examination of the startle response during upper limb stretch perturbations

Unexpected presentation of a startling auditory stimulus (SAS>120 decibels) in a reaction time (RT) paradigm results in the startle reflex and an early release (<100ms) of the preplanned motor response (StartReact effect). Mechanical perturbations applied to the upper limbs elicit short- (M1) and long-latency (M2) stretch reflexes and have also been shown to initiate intended motor responses early (<100ms). Ravichandran et al. (2013) recently proposed that unexpected delivery of a perturbation could also elicit a startle response and therefore the StartReact effect may be responsible for the early trigger of a preplanned response. To investigate this further, we examined startle incidence, RT, and stretch reflex modulation for both expected and unexpected perturbations. In Experiment 1, participants performed active (ACT) and passive (DNI) conditions to an expected large perturbation (similar to previous studies examining M2). The startle response was not observed; however, the perturbation still elicited the voluntary response at short latency (<100ms) and goal-dependent modulation of the M2 response was observed. In Experiment 2, participants performed ACT and DNI conditions to a weak auditory stimulus or a small wrist perturbation. On unexpected trials we probed startle circuitry with a large perturbation or SAS. The SAS consistently elicited a startle response in both ACT and DNI conditions, but startle-like activity was only observed on 17.4% of ACT perturbation probe trials. Our findings suggest that while unexpected upper limb perturbations can be startling, startle triggering of the preplanned voluntary response is not the primary mechanism responsible for goal-dependent modulation of the M2 response.

過酸化物によるピルビン酸キナーゼM2（PKM2）活性制御と酸化ストレス応答機構の解析

過酸化物によるピルビン酸キナーゼM2（PKM2）活性制御と酸化ストレス応答機構の解析

Circulating cell-free DNA indicates M1/M2 responses during septic peritonitis

Circulating cell-free DNA (cfDNA) has been widely suggested as clinical indicator in diseases, including sepsis. It was thought that the cfDNA was coming from the cell lysis, necrosis and apoptosis caused by tissue damages during sepsis. M1 or M2 macrophage-type responses kill or repair in vivo, which is highly relevant with the tissue damages in sepsis. The correlation between cfDNA and M1/M2 responses during sepsis was never investigated. Here, we used bacteria injection induced septic peritonitis mouse model in both M1-dominant C57bl/6 and M2-dominant Balb/c mouse strains. We found that M2-dominant Balb/c mice showed better prognosis of septic peritonitis than C57bl/6 mice, which is corresponded with lower level of cfDNA in septic Balb/c mice compared to septic C57bl/6 mice. By assessing the M1 and M2 related cytokines in both septic Balb/c and C57bl/6 mice, we found out that Balb/c mice has lower tumor necrosis factor α (TNFα) and higher interleukin 10 (IL-10) productions than C57bl/6 mice during septic peritonitis. Especially, when monitoring the monocyte subtypes in peripheral blood of these septic mice, we found out that C57bl/6 showed higher inflammatory (Ly6Chigh) monocyte (corresponding to M1 macrophage) proportion than Balb/c mice. Interestingly, we find out that cfDNA is highly correlated with the ratio of Ly6Chigh monocytes versus Ly6Clow monocytes, which represents M1/M2 (killing/healing) responses. Our study suggested that the cfDNA is a good indicator for evaluating M1/M2 responses in septic peritonitis.

Role of Macrophage (M1 and M2) in Titanium-Dioxide Nanoparticle-Induced Oxidative Stress and Inflammatory Response in Rat.

Titanium-dioxide nanoparticles (TNP) are used in various consumable goods. Evidence has demonstrated the cytotoxicity of TNPs, but exact mechanism is yet to be elucidated. The present study has been aimed at finding out the mechanism of TNP-induced toxicity in biological system. Different doses of anatase-TNPs administrated intravenously to Wistar rats for once a week for 1month and properties of TH cells, macrophages, cytokines secretion, oxidative damage, apoptotic pathway, and hematological and pathological changes were investigated as downstream events of TNP-mediated cytotoxicity. Result suggests that TNPs induce TH1 and TH2 response as measured by immunophenotyping (interferon gamma (IFN-γ) and interleukin (IL)-4) of TH cells, causing induction of M1 (nitric oxide (NO), nitric oxide synthase (iNOS), NF-kappaB (NF-κB), cyclooxygenase-2 (COX-2), IL-1, IL-6, and TNF-α) and M2 (Arg-1, Ym1) macrophages response. At lower dose, TH1 or M1 response counteracted by TH2 or M2 response, resulting in insignificant oxidative damage. However, with increasing dose of TNPs, the M1 response was increased over M2 response resulting in significant tissue damage. The M1-induced inflammatory response was found to cause DNA and chromosomal damage resulting apoptosis induction via upregulation of Bax/Bcl-2 ratio and subsequent loss of mitochondrial membrane potential and cyto c release in splenocytes. The TNP-led inflammatory response also causes damage at different tissue levels.

Modulation of microglia polarization dynamics during diabetic retinopathy in db/db mice

Retinal diseases linked to inflammation are often accompanied by macrophage/microglial cells activation. However, the dynamics between M1 (pro-inflammatory) and M2 (anti-inflammatory) polarization of microglia during diabetic retinopathy (DR) has not been investigated and it might be therapeutically useful. We assessed microglia polarization in retinas from db/db mice and human diabetic donors and also the microglia-mediated anti-inflammatory effects of the bicyclic nojirimycin derivative (1R)-1-dodecylsulfinyl-5N,6O-oxomethylidenenojirimycin (R-DS-ONJ). Visual function in mice was evaluated by electroretinogram (ERG). Expression of pro- and anti-inflammatory markers in the retina was analyzed by immunofluorescence, Western-blot and quantitative real-time PCR. Lipopolysaccharide (LPS)-mediated polarization profile was studied in Bv.2 microglial cells in the absence or presence of anti-inflammatory cytokines (IL4/IL13) or R-DS-ONJ. At 5weeks of age, reduced ERG amplitude values of rod and mixed waves were detected in db/db compared to db/+ mice that correlated with elevated circulating endotoxemia and pro-inflammatory cytokines. At this early stage of DR, the marker of activated microglia Iba-1 co-localized with the M2 marker arginase-1 in the retina. Conversely, in retinas from 8weeks old db/db mice Iba-1-colocalized with active caspase-1, a key component of the inflammasome, reflecting an opposite pattern of microglia polarization. Markers of activated microglia were detected in retinas of diabetic donors. Treatment of Bv.2 cells with LPS and IL4/IL13 or R-DS-ONJ switched the M1 response towards M2. In retinal explants from db/db mice, R-DS-ONJ induced a M2 response. In conclusion, the modulation of microglia polarization dynamics towards a M2 status at early stages of DR offers novel therapeutic interventions.

MiRNA regulation of macrophage inflammatory phenotype

Abstract Classically (M1) and alternatively activated (M2) macrophages play distinct roles in various physiological and disease processes. M1 responses can cause inflammatory disease and tissue damage while M2 macrophages can limit inflammation and promote tissue repair. Therefore, understanding key players driving M1 or M2 phenotype may provide novel targets for therapeutic intervention. MicroRNA are small RNAs that bind to messenger RNA and post-transcriptionally modify gene expression and miR-155 has been associated with inflammatory phenotype. miR-155 was up-regulated more than 100-fold in M1, but not M2 macrophages, and inflammatory genes and proteins that define the M1 phenotype (e.g., iNOS, IL-1b, TNF-a) were reduced up to 72% in miR-155 knockout mouse macrophages. In contrast, miR-155 deficiency did not affect expression of genes associated with M2 macrophages (e.g., Arginase-1). Comparative transcriptional profiling of macrophages derived from wild-type and miR-155 knockout mice revealed that approximately half of the wild-type M1 signature was dependent on miR-155. Among miR-155-dependent M1 inflammatory effectors we found IL-6, iNOS, IL-1b, TNF-a and IL-12 as well as the recently described M1 marker CD38. miR-155 targets Bat5, Mafb, c-Maf, Bach1, Ptprj and Il6ra were increased in miR-155 KO M1 macrophages. Importantly, treatment with a miR-155 oligonucleotide inhibitor suppressed iNOS and TNF-a gene expressions in wild-type M1 macrophages, lending support to the idea that miR-155 targeting may be beneficial in inflammatory disease. Overall, these data indicate that miR-155 plays an essential role in the differentiation and effector potential of inflammatory M1 macrophages.

Differences in gene expression in human monocytes and macrophages from healthy and asthmatic donors in response to IL-4

Abstract The lungs from asthmatics are in a state of Th2-driven inflammation with a shift in macrophage polarization toward an M2 profile induced by IL-4 and IL-13. The number of M2 macrophages in the lung correlates with asthma severity. Women have more lung M2 cells than men and are more likely to have asthma than men. We hypothesized that sex differences affect macrophage polarization in humans. First, we determined that M2-related gene expression was different between healthy and asthmatic donors in response to IL-4 and IL-13. Monocytes from asthmatics expressed more CCL17 in response to IL-4, while CCL22 and MMP12 expression was similar. We found that CD200R was highly expressed in response to IL-4 in monocyte derived-macrophages (MDM) from asthmatics compared with those of healthy donors. Furthermore, this increase was greater in MDM from women compared to men. Moreover, MMP12 and CCL22 expression were not different between the sexes. Interestingly, some genes (TGM2) were only up-regulated in MDM from asthmatics. Second, we analyzed the effect of male and female sex steroid hormones on the IL-4 signaling pathway. In the presence of IL-4, we found that both estrogen (E2) and dihydrotestosterone(active metabolite of testosterone) can induce changes in phosphorylation of Akt but not in STAT6 in MDM. Similar effects on signaling were results were obtained in U937, a human monocytic cell line. The stronger M2 response in female cells could be due to increased expression of estrogen receptors. We found that monocytes from women displayed greater expression of ERa isoforms. These results indicate a potential role for both intrinsic sex differences and E2 as inducers of M2 polarization in macrophages in asthma, leading to worse lung inflammation in women.