Classical Macrophage Activation Research Articles

Classical Activation of Macrophages Leads to Lipid Droplet Formation Without de novo Fatty Acid Synthesis.

Altered lipid metabolism in macrophages is associated with various important inflammatory conditions. Although lipid metabolism is an important target for therapeutic intervention, the metabolic requirement involved in lipid accumulation during pro-inflammatory activation of macrophages remains incompletely characterized. We show here that macrophage activation with IFNγ results in increased aerobic glycolysis, iNOS-dependent inhibition of respiration, and accumulation of triacylglycerol. Surprisingly, metabolite tracing with 13C-labeled glucose revealed that the glucose contributed to the glycerol groups in triacylglycerol (TAG), rather than to de novo synthesis of fatty acids. This is in stark contrast to the otherwise similar metabolism of cancer cells, and previous results obtained in activated macrophages and dendritic cells. Our results establish a novel metabolic pathway whereby glucose provides glycerol to the headgroup of TAG during classical macrophage activation.

Group 2 Innate Lymphoid Cells (ILC2) Suppress Beneficial Type 1 Immune Responses During Pulmonary Cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen preferentially causing disease in immunocompromised individuals such as organ-transplant-recipients, patients receiving immunosuppressive medications or, in particular, individuals suffering from HIV infection. Numerous studies clearly indicated that the control of C. neoformans infections is strongly dependent on a prototypic type 1 immune response and classical macrophage activation, whereas type 2-biased immunity and alternative activation of macrophages has been rather implicated in disease progression and detrimental outcomes. However, little is known about regulatory pathways modulating and balancing immune responses during early phases of pulmonary cryptococcosis. Here, we analyzed the role of group 2 innate lymphoid cells (ILC2s) for the control of C. neoformans infection. Using an intranasal infection model with a highly virulent C. neoformans strain, we found that ILC2 numbers were strongly increased in C. neoformans-infected lungs along with induction of a type 2 response. Mice lacking ILC2s due to conditional deficiency of the transcription factor RAR-related orphan receptor alpha (Rora) displayed a massive downregulation of features of type 2 immunity as reflected by reduced levels of the type 2 signature cytokines IL-4, IL-5, and IL-13 at 14 days post-infection. Moreover, ILC2 deficiency was accompanied with increased type 1 immunity and classical macrophage activation, while the pulmonary numbers of eosinophils and alternatively activated macrophages were reduced in these mice. Importantly, this shift in pulmonary macrophage polarization in ILC2-deficient mice correlated with improved fungal control and prolonged survival of infected mice. Conversely, adoptive transfer of ILC2s was associated with a type 2 bias associated with less efficient anti-fungal immunity in lungs of recipient mice. Collectively, our date indicate a non-redundant role of ILC2 in orchestrating myeloid anti-cryptococcal immune responses toward a disease exacerbating phenotype.

P151 DISRUPTION OF ENDOGENOUS ITACONATE PRODUCTION EXACERBATES EXPERIMENTAL COLITIS

Abstract Background Inflammation is a hallmark of inflammatory bowel disease and alterations in tricarboxylic acid cycle (TCA) metabolism have been identified as major regulators of immune cell phenotype during inflammation and hypoxia. The TCA cycle metabolite, itaconate, is produced by the enzyme aconitate decarboxylase 1 (Acod1) and is highly upregulated during classical macrophage activation and during experimental colitis. Itaconate and cell permeable derivatives have robust anti-inflammatory effects on macrophages, therefore we hypothesized that Acod1-produced itaconate has a protective, anti-inflammatory effect during experimental colitis. Methods and Results Wild type (WT) control and Acod1-/- mice were administered 3% Dextran Sulfate Sodium (DSS) in water for 7 days to induce experimental colitis. After DSS was discontinued, Acod1-/- mice had significantly reduced body weight recovery with increased macroscopic disease severity, and upon dissection had decreased colon length and more severe inflammation. To determine if myeloid cells are the critical Acod1/itaconate-producing cell types, we generated myeloid-specific Acod1 deficient mice, however no differences in weight loss, colon length or inflammatory gene expression were detected compared to WT controls. To test whether supplementation with exogenous itaconate could ameliorate colitis, WT mice were treated with the cell-permeable form of itaconate, dimethyl itaconate (DMI). Administration of DMI significantly improved recovery after 7 days of DSS treatment and significantly reduced inflammatory gene expression in the colon. Conclusion Our data suggest that Acod1-produced itaconate has an important role in the regulation of inflammation during experimental colitis. Although myeloid cells have been thought to be major producers of Acod1 and itaconate, our data indicate that other cell types are involved. These results highlight the importance of this immunometabolic pathway and suggest that preservation or enhancement of this pathway with natural metabolites or metabolite derivatives could have beneficial effects during colitis.

P151 DISRUPTION OF ENDOGENOUS ITACONATE PRODUCTION EXACERBATES EXPERIMENTAL COLITIS

Inflammation is a hallmark of inflammatory bowel disease and alterations in tricarboxylic acid cycle (TCA) metabolism have been identified as major regulators of immune cell phenotype during inflammation and hypoxia. The TCA cycle metabolite, itaconate, is produced by the enzyme aconitate decarboxylase 1 (Acod1) and is highly upregulated during classical macrophage activation and during experimental colitis. Itaconate and cell permeable derivatives have robust anti-inflammatory effects on macrophages, therefore we hypothesized that Acod1-produced itaconate has a protective, anti-inflammatory effect during experimental colitis.

CARD9 Is Required for Classical Macrophage Activation and the Induction of Protective Immunity against Pulmonary Cryptococcosis.

Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Consequently, clinical and animal studies indicate that CARD9 is an important regulator of protective immunity against fungal pathogens. Previous studies suggest that CARD9 is important for the induction of protection against Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system in immunocompromised patients. However, the effect of CARD9 deficiency on the induction of protective immune responses against C. neoformans is unknown. Immunization with a C. neoformans mutant that overexpresses the transcription factor zinc finger 2, denoted LW10, results in protection against an otherwise lethal challenge with wild-type (WT) C. neoformans Our results showed that CARD9 is essential for the induction of vaccine-mediated immunity against C. neoformans infection. We observed significant decreases in interleukin-17 (IL-17) production and significant increases in Th2-type cytokine (IL-4, IL-5, and IL-13) production in CARD9-deficient mice after inoculation with strain LW10. While leukocyte infiltration to the lungs of CARD9-deficient mice was similar in LW10 and WT C. neoformans-infected mice, macrophages derived from CARD9-deficient mice inherently skewed toward an M2 activation phenotype, were unable to contain the growth of LW10, and failed to produce nitric oxide in response to infection with LW10 or stimulation with lipopolysaccharide. These results suggest that CARD9-mediated signaling is required for M1 macrophage activation and fungicidal activity necessary for the induction of vaccine-mediated immunity against C. neoformansIMPORTANCECryptococcus neoformans is a fungal pathogen that is found throughout the environment and can cause life-threatening infections of the lung and central nervous system in severely immunocompromised individuals. Caspase recruitment domain-containing protein 9 (CARD9) is a critical molecule that is activated after interactions of C-type lectin receptors (CLRs) found on the surfaces of specific immune cells, with carbohydrate structures associated with fungi. Patients with defects in CARD9 are significantly more susceptible to a multitude of fungal infections. C. neoformans contains several carbohydrate structures that interact with CLRs on immune cells and activate CARD9. Consequently, these studies evaluated the necessity of CARD9 for the induction of protective immunity against C. neoformans infection. These results are important, as they advance our understanding of cryptococcal pathogenesis and host factors necessary for the induction of protective immunity against C. neoformans.

Aquaporin-1 attenuates macrophage-mediated inflammatory responses by inhibiting p38 mitogen-activated protein kinase activation in lipopolysaccharide-induced acute kidney injury

ObjectiveThis study was designed to investigate the role of AQP1 in the development of LPS-induced AKI and its potential regulatory mechanisms in the inflammatory responses of macrophages.MethodsMale Wistar rats were injected intraperitoneally with LPS, and biochemical and histological renal damage was assessed. The levels of inflammatory mediators, macrophage markers and AQP1 in blood and kidney tissues were assessed by ELISA. RTPCR was used to assess changes in the relative levels of AQP1 mRNA induced by LPS. Western blot and immunofluorescence analyses were performed to assay the activation of the p38 MAPK and NF-κB pathways, respectively. The same detection methods were used in vitro to determine the regulatory mechanisms underlying AQP1 function.ResultsAQP1 mRNA levels were dramatically decreased in AKI rats following the increased expression of inflammatory factors. In vitro experiments demonstrated that silencing the AQP1 gene increased inflammatory mediator secretion, altered the classical activation of macrophages, greatly enhanced the phosphorylation of p38 and accelerated the translocation of NF-κB. Furthermore, these results were blocked by doramapimod, a p38 inhibitor. Therefore, these effects were mediated by the increased phosphorylation of p38 MAPK.ConclusionOur results suggest that altered AQP1 expression may be associated with the development of inflammation in AKI. AQP1 plays a protective role in modulating acute renal injury and can attenuate macrophage-mediated inflammatory responses by downregulating p38 MAPK activity in LPS-induced RAW264.7 cells. The pharmacological targeting of AQP1-mediated p38 MAPK signalling may provide a novel treatment approach for AKI.

The antineoplastic agent anacardic 6-pentadecyl salicylic acid produces immunomodulation in vivo via the activation of MAPKs

Anacardic 6-pentadecyl salicylic acid (6SA) is the active component of Amphipterygium adstringens, a plant used in traditional medicine for the treatment of malaria and vascular diseases and as an anti-bacterial and immune-modulatory agent. However, the effect of 6SA on the immune system remains unclear. In this study, we examined the immune-stimulatory activity of 6SA in 6–8-week-old female BALB/c mice. We found that treatment with 2 mg/kg of 6SA increased the proportions of macrophages after 7 and 14 days of treatment and of natural killer (NK) cells after 14 days of treatment in circulating blood. In lymph nodes, treatment with 6SA for 14 days increased the number of macrophages. In addition, 6SA increases in the systemic levels of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-12, IL-6 and IL-1β and of nitric oxide (NO). We observed an increase in the secretion of Granulocyte/Macrophage Colony Stimulation Factor (GM-CSF) that could explain the increase in the proportion of macrophages. Moreover, 6SA induced the classical activation of macrophages by increasing their expression of MHC-II and their production of TNF-α. These M1-polarised macrophages presented enhanced phagocytosis and NO secretion. This activation was due to induction of the phosphorylation of MAPKs such as ERK, JNK and p38 because specific inhibitors of the phosphorylation of these MAPKs reduced the 6SA-induced phagocytosis and NO and particularly, the secretion of GM-CSF in macrophages by inhibition of ERK. Despite these effects on macrophages, 6SA does not have any direct effect on the proportion of lymphocytes.

IL-16 regulates macrophage polarization as a target gene of mir-145-3p

BackgroundInterleukin 16 is an immunomodulatory chemokine that signals through CD4 + T cells, monocytes, macrophages and dendritic cells. Its expression in immune-related cells enhances the antimicrobial effect and inhibits HIV replication in macrophages. However, the role of IL-16 in macrophage polarization is uncertain. Mir-145 was reported to regulate IL-10 expression by targeting histone deacetylase 11 and promotes alternatively activated macrophage (M2) polarization. Mir-145 was also predicted to target IL-16 mRNA. We aimed to explore the roles of IL-16 and mir-145 in macrophage polarization and antimicrobial functions. MethodsTHP1 monocytes were employed in this study, and their cell activity when incubated with different concentrations of IL-16 was evaluated using the CCK-8 cell counting kit. To obtain polarized macrophages, THP-1 cells were induced by IL-4 and IL-13 following PMA incubation (M2 polarized macrophages) or induced by IFN-gamma and LPS (M1 classical macrophage activation). The influence of IL-16 on macrophage phagocytosis was quantified by the amount of chicken red blood cell phagocytized. IL-16, IL-10 and miR-145 expression in THP1 monocytes and induced macrophages was quantified by quantitative PCR. The miR-145 and IL-16 targeting relationship was verified by the dual luciferase reporter assay. The influence of IL-16 and mir-145 on macrophage polarization was evaluated by M1 and M2 macrophage characterized marker gene expression. ResultsThe M0 macrophage subtype was induced by PMA. The M1 and M2 subtypes of macrophage were successfully induced by M1- and M2-specific induction. M1 macrophages express higher levels of IL-16 than M2 macrophages but express lower levels of IL-10 and mir-145 than M2 cells. IL-16 with a concentration up to 150 ng/mL has no influence on THP-1 cell proliferation but improves macrophage phagocytosis ability with the down-expression of IL-10 and up-expression of pro-inflammatory cytokines such as IL-1a and IL-6. Knockdown with its target siRNA is beneficial for macrophage maintenance but reduces phagocytosis ability. Mir-145 specifically targets the IL-16 3’UTR verified by the dual luciferase reporter assay. Mir-145 downregulates IL-16 expression and upregulates IL-10 expression, thereby promoting M2 macrophage polarization. ConclusionIL-16 modulates macrophage polarization through regulating IL-10, IL-1a and IL-6 expression. Mir-145 is involved in M2 macrophage polarization by targeting IL-16 and enhancing IL-10 expression.

Modulation of Macrophage Polarization by Human Glomerular Mesangial Cells in Response to the Stimuli in Renal Microenvironment.

Mesangial cell (MC) activation and macrophage infiltration are 2 major events closely related with each other in mesangial proliferative glomerulonephritis. In the anti-Thy 1 nephritis model, macrophages mediate the damage and also the expansion of mesangium through secreting various inflammatory factors; however, in glomerular microenvironment how MCs affect macrophage activity in the presence of various stimuli have not yet been understood. In the present study, we found that resting human MCs (HMCs) constitutively expressed chemokine [C-C motif] ligand 2 (CCL-2) and interleukin (IL)-6 and induced M2 polarization of macrophages in the coculture system. HMC proliferation and migration and expression of IL-6, CCL-2, and macrophage colony-stimulating factor in HMCs were enhanced after platelet-derived growth factor (PDGF)-BB stimulation, among which CCL-2 was responsible for inducing the M2 polarization of macrophages. Furthermore, PDGF-BB-stimulated HMCs alleviated the classical activation of macrophages and drove more intensified M2 polarization of macrophages than resting HMCs did. However, lipopolysaccharide and interferon-γ (IFN-γ) stimulated HMCs maintained the M1 phenotype of cocultured macrophages. In conclusion, MCs actively participated in glomerular inflammation through influencing macrophage polarization. The interplay between MCs and infiltrated macrophages is finely modulated by secretory factors such as PDGF-BB and IFN-γ in response to the renal inflammatory microenvironment.

Autocrine IL-10 Signaling Promotes Dendritic Cell Type-2 Activation and Persistence of Murine Cryptococcal Lung Infection.

The substantial morbidity and mortality caused by invasive fungal pathogens, including Cryptococcus neoformans, necessitates increased understanding of protective immune responses against these infections. Our previous work using murine models of cryptococcal lung infection demonstrated that dendritic cells (DCs) orchestrate critical transitions from innate to adaptive immunity and that IL-10 signaling blockade improves fungal clearance. To further understand interrelationships among IL-10 production, fungal clearance, and the effect of IL-10 on lung DCs, we performed a comparative temporal analysis of cryptococcal lung infection in wild type C57BL/6J mice (designated IL-10+/+) and IL-10-/- mice inoculated intratracheally with C. neoformans (strain 52D). Early and sustained IL-10 production by lung leukocytes was associated with persistent infection in IL-10+/+ mice, whereas fungal clearance was improved in IL-10-/- mice during the late adaptive phase of infection. Numbers of monocyte-derived DCs, T cells, and alveolar and exudate macrophages were increased in lungs of IL-10-/- versus IL-10+/+ mice concurrent with evidence of enhanced DC type-1, Th1/Th17 CD4 cell, and classical macrophage activation. Bone marrow-derived DCs stimulated with cryptococcal mannoproteins, a component of the fungal capsule, upregulated expression of IL-10 and IL-10R, which promoted DC type-2 activation in an autocrine manner. Thus, our findings implicate fungus-triggered autocrine IL-10 signaling and DC type-2 activation as important contributors to the development of nonprotective immune effector responses, which characterize persistent cryptococcal lung infection. Collectively, this study informs and strengthens the rationale for IL-10 signaling blockade as a novel treatment for fungal infections.

NFATc3 deficiency reduces the classical activation of adipose tissue macrophages

Nuclear factors of activated T cells (NFAT) c3 have a prominent role in the regulation of proinflammatory factors in immune cells. The classically activated M1 macrophages are key players in the initiation and maintenance of adipose tissue (AT) inflammation. The role of NFATc3 in obesity and AT inflammation is unknown. We set out to determine how deficiency of NFATc3 effected macrophage polarization, inflammation and insulin resistance in visceral AT of high-fat diet (HFD)-fed mice. Nfatc3−/− and WT mice were fed a HFD for 8–17 weeks. Epididymal white AT (eWAT) F4/80(+) cells were characterized by fluorescence-activated cell sorting and quantitative RT-PCR. Results showed that Nfatc3−/− mice developed HFD-induced obesity similar to WT mice, but insulin sensitivity and glucose tolerance were improved, and liver fat accumulation was reduced in Nfatc3−/− mice compared to WT control mice. Moreover, M1 macrophage content and proinflammatory factors were reduced, whereas the alternatively activated M2 macrophage content was increased in eWAT of HFD-fed Nfatc3−/− mice compared to that of WT mice. In addition, eWAT insulin signaling was improved in HFD-fed Nfatc3−/− mice. Importantly, after bone-marrow-derived macrophages had been isolated from Nfatc3−/− mice and cultured in vitro, treatment of these cells with interferon-γ and lipopolysaccharide resulted in reduction of M1 inflammatory markers, suggesting that NFATc3 promoted M1 polarization by a cell-autonomous mechanism. The results demonstrated that NFATc3 played an important role in M1 macrophage polarization, AT inflammation and insulin resistance in response to obesity through transcriptional activation of proinflammatory genes.

Polysaccharide rich extract (PRE) from Tinospora cordifolia inhibits the intracellular survival of drug resistant strains of Mycobacterium tuberculosis in macrophages by nitric oxide induction

Plethora of clinical and scientific information obtained in recent past has strengthened the idea that targeting critical constituents of host immune system may have beneficial outcomes for the treatment of tuberculosis. Macrophages being the primary host for Mycobacterium tuberculosis, offer an attractive target for modulation. Owing to their negligible toxicity, plant derived polysaccharides with the ability to activate macrophages; are suitable candidates for immunomodulation. In the present study, effects of polysaccharide rich extract (PRE) isolated from Tinospora cordifolia, on the survival of intracellular MTB strains and activation of macrophages were investigated. PRE treatment up regulated the expression of pro-inflammatory cytokines such as IL-β, TNF-α, IL-6, IL-12, and IFN-γ in RAW 264.7 cell line. Up regulation in the expression of NOS2 was observed along with concomitant enhanced nitric oxide production post PRE treatment. Surface expression of MHC-II and CD-86 was up regulated after PRE treatment. Above results suggested the classical activation of macrophages by PRE treatment. Furthermore, PRE treatment led to the activation of all the three classes of MAPK i.e p38, ERK and JNK MAPKs. Further, PRE up regulated the expression of cytokines, NOS-2, MHC-II and CD-86 in MTB infected macrophages. PRE treatment inhibited the intracellular survival of drug resistant MTB in macrophages which was partially attributed to PRE mediated NO induction. Thus our data demonstrate classical activation of macrophages by PRE treatment and killing of intracellular MTB by NO induction.

Abstract P296: High Salt (NaCl) Induces Metabolic Modifications and Affects Classic and Alternative Macrophage Activation

High intake of dietary sodium is a risk factor for cardiovascular disease. Previously, we have shown that an increase in extracellular hypertonic sodium (+40 mM Na + ; HS) inhibits alternatively-activated M2 macrophage (M(IL4+IL13)) gene signature and function, while boosting classic M1 macrophage polarization (M(LPS)) and bacterial killing capacity. M1 macrophages predominantly rely on glycolysis, whereas M2 macrophages use oxidative phosphorylation, fueled by the oxidation of fatty acids, at a late phase of activation (24 h). The aim of this study was to elucidate the role of HS on the metabolic signature of M1 and M2 macrophages. Therefore, we quantified 13 C incorporation into intermediates of central carbon metabolism via GC-MS based pulsed stable isotope resolved metabolomics (pSIRM), and performed Seahorse analyses at varying time points during M1/M2-activation. After 24 h, LPS-induced increase in basal ECAR (extracellular acidification as surrogate parameter for glycolysis) and lactate production were further boosted under HS compared to isotonic salt conditions (NS), whereas mitochondrial respiration (OCR) was not affected. On the other hand, IL4+IL13-induced increase in OCR was decreased under HS compared to NS, while ECAR was not affected at this stage. Regarding TCA-cycle, we have found an early (1 h and 3 h post-stimulation) increase in citrate levels in M1 and M2 macrophages independent of HS, which further increased after 24 h in M1 and M1+HS. Interestingly, both in early and late phase of activation, glucose-derived 13 C incorporation into citrate was lower in HS-treated compared to NS-treated M0, M1 and M2 macrophages, suggesting an alternative replenishing source. Consistently, other TCA-cycle intermediates increased after 24h (especially in M1 and even further in M1+HS), whereas glucose-derived 13 C incorporation was minimal. Our data suggests that under hypertonic sodium macrophages re-direct TCA-cycle intermediates into the production of pro-inflammatory mediators (as described for late-phase M1 macrophages), whereas TCA-cycle itself is replenished by other alternative metabolic substrates. These HS-induced metabolic modifications might play an important role for macrophage activation and function.

Graphene oxide nanosheets increase Candida albicans killing by pro-inflammatory and reparative peritoneal macrophages.

Graphene oxide (GO) is a new nanomaterial with different potential biomedical applications due to its excellent physicochemical properties and ease of surface functionalization. Macrophages play key roles in the control of fungal infections preventing invasive candidiasis by both limiting the growth of the opportunistic fungal pathogen Candida albicans and activating other immune effector cells. In order to know if macrophages maintain their immunocompetence against this microorganism after GO uptake, we have evaluated the interactions at the interface of GO nanosheets, macrophages and Candida albicans. Poly (ethylene glycol-amine)-derivatized GO nanosheets labelled with fluorescein isothiocyanate (FITC-PEG-GO), were efficiently taken up by peritoneal macrophages inducing a significant increase of C. albicans phagocytosis by both pro-inflammatory macrophages (M1/stimulated with LPS/IFN-γ) and reparative macrophages (M2/stimulated with IL-4). On the other hand, after FITC-PEG-GO treatment and C. albicans infection, the percentages of GO+ macrophages diminished when Candida uptake increased in every condition (macrophages with no stimuli, M1 and M2 macrophages), thus suggesting the exocytosis of this nanomaterial as a dynamic mechanism favoring fungal phagocytosis. For the first time, we have analyzed the effects of PEG-GO nanosheets on Candida albicans killing by unstimulated, M1 and M2 macrophages, evidencing that intracellular GO modulates the macrophage candidacidal activity in a multiplicity of infection (MOI) dependent manner. At MOI 1, the high intracellular GO levels increase the fungicidal activity of basal and stimulated macrophages. At MOI 5, as intracellular GO decreases, the previous pro-inflammatory or reparative stimulus predefines the killing ability of macrophages. In summary, GO treatment enhances classical M1 macrophage activation, important for pathogen eradication, and diminishes alternative activation of M2 macrophages, thus decreasing fungal persistence and avoiding chronic infectious diseases.

C-terminal truncation of IFN-\u03b3 inhibits proinflammatory macrophage responses and is deficient in autoimmune disease

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages. In acute peritonitis this signature was absent in Mmp12–/– mice and recapitulated in Mmp12+/+ mice treated with a MMP12-specific inhibitor. Similarly, loss-of-MMP12 increases IFN-γ–dependent proinflammatory markers and iNOS+/MHC class II+ macrophage accumulation with worse lymphadenopathy, arthritic synovitis and lupus glomerulonephritis. In active human systemic lupus erythematosus, MMP12 levels were lower and IFN-γ higher compared to treated patients or healthy individuals. Hence, macrophage proteolytic truncation of IFN-γ attenuates classical activation of macrophages as a prelude for resolving inflammation.

The Kat in the HAT: The Histone Acetyl Transferase Kat6b (MYST4) Is Downregulated in Murine Macrophages in Response to LPS.

Epigenetic modulators, including histone methylases, demethylases, and deacetylases, have been implicated previously in the regulation of classical and alternative macrophage activation pathways. In this study, we show that the histone acetyl transferase (HAT) Kat6B (MYST4) is strongly suppressed (>80%) in macrophages by lipopolysaccharide (LPS) (M1 activation), while Kat6A, its partner in the MOZ/MORF complex, is reciprocally upregulated. This pattern of expression is not altered by LPS together with the adenosine receptor agonist NECA (M2d activation). This is despite the observation that miR-487b, a putative regulator of Kat6B expression, is mildly stimulated by LPS, but strongly suppressed by LPS/NECA. Other members of the MYST family of HATs (Kat5, Kat7, and Kat8) are unaffected by LPS treatment. Using the pLightswitch 3′UTR reporter plasmid, the miR-487b binding site in the Kat6b 3′UTR was found to play a role in the LPS-mediated suppression of Kat6B expression, but other as-yet unidentified factors are also involved. As Kat6B is a HAT that has the potential to modulate gene expression by its effects on chromatin accessibility, we are continuing our studies into the potential roles of this epigenetic modulator in macrophage activation pathways.

Regular exercise improves inflammatory responses by resident or recruited macrophages against bacterial pathogens

Regular, moderate-intensity exercise is beneficial to host defenses against infections. Such exercise regimes improve the inflammatory and bactericidal potencies of macrophages, cells that are crucial in activating both innate and adaptive immune responses. The M1 type (classical) macrophage activation after recognition of the pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), induces pro-inflammatory responses in infected foci; these responses are necessary to effectively clear the pathogens. We reported previously that a 3-week-long treadmill exercise program enhanced LPS-induced interleukin (IL)‑12 mRNA expression and IL-12 secretion by downregulating β 2 -adrenergic receptor expression in the murine peritoneal resident macrophages, suggesting that regular exercise promotes the Toll-like receptor signaling and expression of its target genes in resident macrophages. We also reported that an 8-week-long, voluntary wheel-running exercise program potentiated LPS-induced IL-1β and IL-18 secretion without affecting the intracellular pro-IL-1β or pro-IL-18 mRNA or protein levels in the thioglycollate-elicited peritoneal murine macrophages. This program increased the intracellular caspase-1 protein levels. These results suggest that regular exercise promotes pro-IL-1β and pro-IL-18 maturation by increasing the levels of the inflammasome-activated caspase-1 in the recruited macrophages. Here, we review our collective studies and compare the mechanistic differences by which regular exercise improves pro-inflammatory responses to bacterial pathogens by resident or recruited macrophages.

PD-1 blockade activates conventional CD4 T cells and the innate immune response during glioblastoma eradication

Abstract Blockade of immune cell co-inhibitory receptor PD-1 using monoclonal antibodies enables anti-tumor immune responses in various solid tumors and lymphoid malignancies. Our laboratory previously demonstrated PD-1 blockade elicits an anti-tumor immune response resulting in tumor rejection and long-term survival in approximately 50% of mice with an orthotopic GL261 glioblastoma, despite lacking a corresponding accumulation of CD8+ cytotoxic T cell in the tumor or draining lymph nodes. In this investigation, we evaluated the role of conventional CD4+ T cells and the innate immune response in PD-1 mediated anti-glioma immunity using multiplex technologies for immunohistochemistry and flow cytometry. In response to PD-1 monotherapy, intratumoral CD4 T cells expressed significantly elevated levels of proteins required for T cell proliferation, activation, and effector function. CD4 T cell activation was accompanied by the classical activation and M1 polarization of resident microglia and tumor-infiltrating macrophages, including down-regulation of PD-L1 and up-regulation of MHC class II surface expression. We also demonstrated that depletion of either CD4 or CD8 T cells was sufficient to completely ablate anti-PD-1-mediated tumor eradication and long-term survival. Our data suggests CD4 T cells and myeloid cells may play a prominent role in the eradication of glioblastoma by PD-1 blockade.

Type 2 macrophages and Th2 CD4+ cells in interstitial lung diseases (ILDs): an overview.

Interstitial lung diseases (ILDs) are a heterogeneous group characterized mainly by damage to pulmonary parenchyma, through histopathological processes such as granulomatous pneumopathy, inflammation and fibrosis. Factors that generate susceptibility to ILDs include age, exposure to occupational and environmental compounds, genetic, family history, radiation and chemotherapy/immunomodulatory and cigarette smoke. IFN-γ, IL-1β, and LPS are necessary to induce a classical activation of macrophages, whereas cytokines as IL-4 and IL-13 can induce an alternative activation in macrophages, through the JAK-STAT mediated signal transduction. M2 macrophages are identified based on the gene transcription or protein expression of a set of M2 markers. These markers include transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. Fibrotic lung disorders may have a M2 polarization background. The Th2 pathway with an elevated CCL-18 (marker of M2) concentration in the bronchoalveolar lavage fluid (BALF) is linked to fibrosis in ILDs. Besides the role of M2 in tissue repair and ECM remodeling, activated fibroblasts summon and stimulate macrophages by producing MCP-1, M-CSF and other chemokines, as well as activated macrophages secrete cytokines that attract and stimulate proliferation, survival and migration of fibroblast mediated by platelet-derived growth factor (PDGF). (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 98-108).

Interferon Gamma Induces Reversible Metabolic Reprogramming of M1 Macrophages to Sustain Cell Viability and Pro-Inflammatory Activity

Classical activation of M1 macrophages with lipopolysaccharide (LPS) is associated with a metabolic switch from oxidative phosphorylation to glycolysis. However, the generalizability of such metabolic remodeling to other modes of M1 macrophage stimulation, e.g. type II interferons (IFNs) such as IFNγ, has remained unknown as has the functional significance of aerobic glycolysis during macrophage activation. Here we demonstrate that IFNγ induces a rapid activation of aerobic glycolysis followed by a reduction in oxidative phosphorylation in M1 macrophages. Elevated glycolytic flux sustains cell viability and inflammatory activity, while limiting reliance on mitochondrial oxidative metabolism. Adenosine triphosphate (ATP) distributed by aerobic glycolysis is critical for sustaining IFN-γ triggered JAK (Janus tyrosine kinase)-STAT-1 (Signal Transducer and Activator of Transcription 1) signaling with phosphorylation of the transcription factor STAT-1 as its signature trait. Inhibition of aerobic glycolysis not only blocks the M1 phenotype and pro-inflammatory cytokine/chemokine production in murine macrophages and also human monocytes/macrophages. These findings extend on the potential functional role of immuno-metabolism from LPS- to IFNγ-linked diseases such as atherosclerosis and autoimmune disease.