Lipid homeostasis and inflammatory activation are disturbed in classically activated macrophages with peroxisomal β‐oxidation deficiency

Macrophage activation is characterized by pronounced metabolic adaptation. Classically activated macrophages show decreased rates of mitochondrial fatty acid oxidation and oxidative phosphorylation and acquire a glycolytic state together with their pro‐inflammatory phenotype. In contrast, alternatively activated macrophages require oxidative phosphorylation and mitochondrial fatty acid oxidation for their anti‐inflammatory function. Although it is evident that mitochondrial metabolism is regulated during macrophage polarization and essential for macrophage function, little is known on the regulation and role of peroxisomal β‐oxidation during macrophage activation. In this study, we show that peroxisomal β‐oxidation is strongly decreased in classically activated bone‐marrow‐derived macrophages (BMDM) and mildly induced in alternatively activated BMDM. To examine the role of peroxisomal β‐oxidation in macrophages, we used Mfp2^?/? BMDM lacking the key enzyme of this pathway. Impairment of peroxisomal β‐oxidation in Mfp2^?/? BMDM did not cause lipid accumulation but rather an altered distribution of lipid species with very‐long‐chain fatty acids accumulating in the triglyceride and phospholipid fraction. These lipid alterations in Mfp2^?/? macrophages led to decreased inflammatory activation of Mfp2^?/? BMDM and peritoneal macrophages evidenced by impaired production of several inflammatory cytokines and chemokines, but did not affect anti‐inflammatory polarization. The disturbed inflammatory responses of Mfp2^?/? macrophages did not affect immune cell infiltration, as mice with selective elimination of MFP2 from myeloid cells showed normal monocyte and neutrophil influx upon challenge with zymosan. Together, these data demonstrate that peroxisomal β‐oxidation is involved in fine‐tuning the phenotype of macrophages, probably by influencing the dynamic lipid profile during macrophage polarization.     

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBP^chr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11^−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.     

CD46 accelerates macrophage‐mediated host susceptibility to meningococcal sepsis in a murine model

CD46, a membrane cofactor expressed on all nucleated human cells, plays an essential role in suppressing autoimmune reactions and protecting host cells from complement‐mediated attack. Human transgenic CD46 homozygous mice (CD46^+/+) are prone to lethal sepsis upon infection with Neisseria meningitidis (N. meningitidis). However, the underlying mechanisms are poorly understood. Here, we determined thatCD46^+/+ mice produce large numbers of M1 type macrophages with enhanced surface expression of MHC II and production of pro‐inflammatory mediators such as IL‐6, TNF, IL‐12, and IL‐1β In the presence of M‐CSF or GM‐CSF, CD46 signaling enhances monocyte‐macrophage differentiation. Additionally, CD46^+/+ macrophages rapidly undergo apoptosis upon LPS challenge or meningococcal infection, which could contribute to uncontrolled bacterial dissemination in vivo. Adoptive transfer of CD46^+/+ peritoneal macrophages aggravated septic responses in wild‐type mice, but the depletion of macrophages partially alleviated septic reactions in CD46^+/+ mice after N. meningitidis infection. Our findings reveal a novel role of CD46 in accelerating inflammatory responses upon meningococcal infection or LPS stimulation by regulating the functional polarization and survival of macrophages.     

Hematopoietic plakophilin‐3 regulates acute tissue‐specific and systemic inflammation in mice

Plakophilin‐3 (PKP3) is a member of the armadillo protein family, which is important in cell?cell contacts and signaling during development and tumorigenesis. In conventional facilities, PKP3‐deficient mice (PKP3^?/?) develop spontaneous dermatitis, indicating a possible involvement of PKP3 in inflammatory responses. Here, we show that PKP3 deficiency sensitizes mice to irritant contact dermatitis induced by phorbol myristate acetate (PMA). This sensitization occurred in mice with PKP3 deficiency in the hematopoietic system (PKP3^?/?hem), but not if the deficiency was specific to skin keratinocytes (PKP3^?/?ker). In a model of dextran sulfate sodium induced colitis, ubiquitous PKP3 deletion, but not intestinal epithelial PKP3 deficiency (PKP3^?/?IEC), impaired survival from disease. Interestingly, PKP3^?/?hem mice also displayed increased sensitivity to dextran sulfate sodium induced colitis. Finally, PKP3^?/? mice were more sensitive to the lethality of lipopolysaccharide (LPS) injection than wild‐type (WT) mice, and this phenotype was associated with increased intestinal permeability. PKP3^?/?IEC mice did not reproduce the enhanced endotoxin reactivity of PKP3^?/? mice, in contrast to PKP3^?/?hem mice. Finally, in vitro stimulation of WT neutrophils with LPS or PMA increased Pkp3 expression. In conclusion, our data highlight a novel role for hematopoietic PKP3 in the regulation of both locally and systemically induced immune responses. Nonetheless, further research is needed to unravel the underlying mechanism.     

Chemokine receptor Ccr5 deficiency induces alternative macrophage activation and improves long‐term renal allograft outcome

The chemokine (C‐C motif) receptor 5 (CCR5) has been implicated in experimental and clinical allograft rejection. To dissect the function of CCR5 in acute and chronic renal allograft rejection, bilaterally nephrectomized WT and Ccr5^?/? C57BL/6 mice were used as recipients of WT BALB/c renal allografts and analyzed 7 and 42 days after transplantation. Lesion scores (glomerular damage, vascular rejection, tubulointerstitial inflammation) and numbers of CD4⁺, CD8⁺, CD11c⁺ and alpha smooth muscle actin (αSMA)⁺ cells were reduced in allografts from Ccr5^?/? recipients during the chronic phase. Increasing creatinine levels indicated deterioration of allograft function over time. While mRNA expression of Th1‐associated markers decreased between 7 and 42 days, Th2‐associated markers increased. Markers for alternatively activated macrophages (arginase 1, chitinase 3‐like 3, resistin‐like α, mannose receptor, C type 1), were strongly upregulated (mRNA and/or protein level) only in allografts from Ccr5^?/? recipients at 42 days. Ccr5 deficiency shifted intragraft immune responses during the chronic phase towards the Th2 type and led to accumulation of alternatively activated macrophages. Additionally, splenocytes from unchallenged Ccr5^?/? mice showed significantly increased arginase 1 and mannose receptor 1 mRNA levels, suggesting constitutive alternative activation of splenic macrophages. We conclude that Ccr5 deficiency favors alternative macrophage activation. This finding may be relevant for other inflammatory diseases that involve macrophage activation and may also influence future therapeutic strategies targeting CCR5.     

p21waf1/CIP1, a CDK inhibitor and a negative feedback system that controls macrophage activation

p21^WAF1/CIP1 (p21) is a crucial CDK inhibitor that controls the cell cycle. This molecule is also involved in the regulation of apoptosis and gene expression. However, like many other cell regulators, the functional activity of p21 depends on its cellular context and is controlled through phosphorylation and protein–protein interactions. p21 is also important in cells of the immune system regulating the cell cycle and preventing apoptosis of macrophages. In this issue of the European Journal of Immunology, two reports investigate the role of p21 further determining its critical role as a negative regulator of macrophage activation, in particular inhibiting the LPS‐dependent induction of TNF‐α and IL‐1β. The inhibition mediated by p21 is shown to be related to NF‐κB activity. Furthermore, the observation that p21^?/? mice are more susceptible to septic shock supports the notion that p21 is a negative regulator of macrophage activation and therefore a potential new target to control inflammatory diseases.     

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin‐induced pulmonary fibrosis

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78^+/?, Chop^?/? and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78^+/? mice were strongly protected from bleomycin‐induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL‐positive cells were observed in Grp78^+/? mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78^+/? mice was also strongly positive for cleaved caspase‐3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop^?/? mice resulted in increased quasi‐static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase‐1‐positive macrophages that were negative for cleaved caspase‐3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78‐ and CHOP‐mediated macrophage apoptosis was found to protect against bleomycin‐induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78‐mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NTPDase1 governs P2X7‐dependent functions in murine macrophages

P2X₇ receptor is an adenosine triphosphate (ATP)‐gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X₇ effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X₇‐dependent responses in these cells. Macrophages isolated from NTPDase1‐null mice (Entpd1^?/?) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1^+/+). Entpd1^?/? macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL‐1β and IL‐18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo‐Pro‐1 more efficiently (suggestive of increased pore formation) than Entpd1^+/+ cells. Consistent with these observations, NTPDase1 regulated P2X₇‐associated IL‐1β release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase‐1. NTPDase1 also inhibited IL‐1β release in vivo in the air pouch inflammatory model. Exudates of LPS‐injected Entpd1^?/? mice had significantly higher IL‐1β levels when compared with Entpd1^+/+ mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X₇‐dependent macrophage responses.     

Alterations to chromatin in intestinal macrophages link IL‐10 deficiency to inappropriate inflammatory responses

Intestinal macrophages (IMs) are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL‐10 maintains the macrophage anti‐inflammatory response such that loss of IL‐10 results in chronic intestinal inflammation. To investigate how IL‐10‐deficiency alters IM programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone‐marrow, from both wild‐type and IL‐10‐deficient (Il10^?/?) mice. We identified chromatin accessibility changes associated with bacterial exposure and IL‐10 deficiency in both bone marrow derived macrophages and IMs. Surprisingly, Il10^?/? IMs adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, when recombinant IL‐10 was added to Il10^?/? cells, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL‐10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model in which IL‐10‐deficiency leads to chromatin alterations that contribute to a loss of IM tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation.     

An optimized Protocol for Human M2 Macrophages using M‐CSF and IL‐4/IL‐10/TGF‐β Yields a Dominant Immunosuppressive Phenotype

Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. We investigated and compared a panel of polarization protocols of blood‐derived monocytes to achieve a stable, optimal and effective regimen for in vitro induction of immunosuppressive human macrophages, evaluating their surface receptor expression, cytokine profile, scavenging function and ability to suppress T‐cell proliferation. Importantly, we assessed the effect of copolarization or secondary pro‐inflammatory stimulation of a primary anti‐inflammatory activation phenotype. A combination of IL‐4/IL‐10/TGF‐β yielded a relatively stable and dominant immunosuppressive phenotype characterized by higher IL‐10 production and down‐regulated TNF‐α, IL‐6, CD86, CD274 and MHC II expression. Functionally, IL‐4/IL‐10/TGF‐β‐stimulated macrophages (M2) had a potent deactivating effect on a subsequent pro‐inflammatory LPS/IFNγ‐activated macrophage (M1) stimulation and significantly suppressed T‐cell proliferation. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti‐inflammatory using this protocol. Pre‐differentiation with GM‐CSF or M‐CSF was further demonstrated to enhance final M1/M2 activation status. Our findings indicate a robust polarization protocol for generation of specific immunosuppressive human monocyte‐derived macrophages.     

Local effects of human PCSK9 on the atherosclerotic lesion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low‐density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE^?/? and LDLR^?/? mice, which were then placed on a high‐fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow‐derived PCSK9 progressively accumulated in lesions of apoE^?/? recipient mice, while increasing the infiltration of Ly6C^hi inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b‐ and Ly6C^hi‐positive cell numbers in spleens of apoE^?/? mice. In vitro, expression of hPCSK9 in LPS‐stimulated macrophages increased mRNA levels of the pro‐inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti‐inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR‐dependent, as PCSK9 protein was not detected in lesions of LDLR^?/? recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR‐dependent but cholesterol‐independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The CD20 homolog Ms4a8a integrates pro‐ and anti‐inflammatory signals in novel M2‐like macrophages and is expressed in parasite infection

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor‐associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a⁺ macrophages in M2‐associated infectious pathologies. In late‐stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll‐like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL‐4). LPS/dexamethasone/IL‐4‐induced Ms4a8a⁺ BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF‐κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL‐4‐stimulated Ms4a8a⁺ BMDMs and in peritoneal macrophages from late‐stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2‐like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.     

Epstein–Barr virus‐induced gene 3 suppresses T helper type 1, type 17 and type 2 immune responses after Trypanosoma cruzi infection and inhibits parasite replication by interfering with alternative macrophage activation

The Epstein–Barr virus‐induced gene 3 (EBI3) is a member of the interleukin‐12 (IL)‐12) family structurally related to the subunit p40 of IL‐12 and forms a heterodimer either with the p28 subunit to build IL‐27 or with p35 to form IL‐35. Interleukin‐27 is secreted by antigen‐presenting cells whereas IL‐35 appears to be produced mainly by regulatory T cells and regulatory B cells but both cytokines negatively regulate inflammatory immune responses. We here analysed the function of EBI3 during infection with the intracellular parasite Trypanosoma cruzi. Compared with C57BL/6 wild‐type mice, EBI3‐deficient (EBI3^?/?) mice showed a higher parasitaemia associated with an increased mortality rate. The EBI3^?/? mice displayed an elevated inflammatory immune response with an increased production of T helper type 1 (Th1‐), Th2‐ and Th17‐derived cytokines. The increased Th2 immune response appears to have over‐ridden the otherwise protective Th1 and Th17 immune responses by the induction of arginase‐1‐expressing alternatively activated macrophages in these mice. Hence, neutralization of IL‐4 and arginase‐1 activity partially restored protective immune responses in EBI3^?/? mice. So far, our results demonstrate that EBI3 is an essential general regulator of inflammatory immune responses in experimental Chagas disease and is required for control of T. cruzi infection by inhibiting Th2‐dependent alternative macrophage activation. Further studies are needed to dissect the underlying mechanisms and clarify whether EBI3 association with IL‐27 or/and IL‐35 accounts for its anti‐inflammatory character in parasitic disease.     

The CDK domain of p21 is a suppressor of IL‐1β‐mediated inflammation in activated macrophages

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21^(WAF1/CIP1), an established suppressor of cell cycle progression, is a inhibitor of IL‐1β synthesis in macrophages. Mice deficient in p21 (p21^?/?) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL‐1β. Administration of IL‐1 receptor antagonist reduces LPS‐induced lethality in p21^?/? mice. Analysis of isolated macrophages, which are one of the central producers of IL‐1β, reveals that deficiency for p21 led to more IL‐1β mRNA and pro‐protein synthesis following TLR ligation. The increase in IL‐1β pro‐protein is associated with elevated secretion of active IL‐1β by p21^?/? macrophages. siRNA‐mediated knockdown of p21 in human macrophages results in increased IL‐1β secretion as well. A peptide mapping strategy shows that the cyclin‐dependent‐kinase (CDK)‐binding domain of p21 is sufficient to reduce the secretion of IL‐1β by p21^?/? macrophages. These data suggest a novel role for p21 and specifically for the CDK‐binding domain of p21^(WAF1/CIP1) in inhibiting inflammation.     

Monocytes/macrophages infected with Toxoplasma gondii do not increase co‐stimulatory molecules while maintaining their migratory ability

Toxoplasma gondii is an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells and use them for dispersal throughout the body, but the activation state of those cells is unknown. We investigated the ability of human and murine cells from monocytic/macrophage lineages that had not previously been exposed to inflammatory cytokines to up‐regulate co‐stimulatory and adhesion molecules upon infection. Toxoplasma gondii‐infected human monocytes (freshly isolated and THP1 lineage) were unable to up‐regulate CD86, CD83, CD40 or CD1a. CD80 expression increased in infected cells but expression of l ‐selectin and β2 integrin was unaltered. We evaluated the ability of infected macrophages from wild type C57/BL/6 or CD14^?/? mice to migrate in 8 μm transwells. Infected cells from CD14^?/? mice were more likely to de‐adhere than infected cells from wild type mice but they did not show any increase in migratory ability. The non‐stimulatory profile of these infected cells may contribute to parasite spread throughout the lymphatic circulation in the initial phases of infection.     

Activation of murine invariant NKT cells promotes susceptibility to candidiasis by IL‐10 induced modulation of phagocyte antifungal activity

Invariant NKT (iNKT) cells play an important role in a variety of antimicrobial immune responses due to their ability to produce high levels of immune‐modulating cytokines. Here, we investigated the role of iNKT cells in host defense against candidiasis using Jα18‐deficient mice (Jα18^?/?), which lack iNKT cells. Jα18^?/? mice were more resistant to the development of lethal candidiasis than wild‐type (WT) mice. In contrast, treatment of WT mice with the iNKT cell activating ligand α‐galactosylceramide markedly enhanced their mortality after infection with Candida albicans. Serum IL‐10 levels were significantly elevated in WT mice in response to infection with C. albicans. Futhermore, IL‐10 production increased after in vitro coculture of peritoneal macrophages with iNKT cells and C. albicans. The numbers of peritoneal macrophages, the production of IL‐1β and IL‐18, and caspase‐1 activity were also significantly elevated in Jα18^?/? mice after infection with C. albicans. The adoptive transfer of iNKT cells or exogenous administration of IL‐10 into Jα18^?/? reversed susceptibility to candidiasis to the level of WT mice. These results suggest that activation of iNKT cells increases the initial severity of C. albicans infection, most likely mediated by IL‐10 induced modulation of macrophage antifungal activity.