Characterization of ST2 transgenic mice with resistance to IL‐33

IL‐33, a member of the IL‐1 family, activates MAPK and NF‐κB through its receptor ST2L and IL‐1RAcP. ST2, a member of the IL‐1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL‐33 and to inhibit the IL‐33/ST2L/IL‐1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL‐33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal MΦ, splenomegaly, accumulation of periodic acid‐Schiff‐positive material in the lung, and high concentrations of serum IL‐5 and IL‐13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal MΦ from IL‐33‐stimulated mice, mRNA expression of M2 MΦ marker genes were increased compared with thioglycollate‐elicited peritoneal MΦ. The IL‐33‐stimulation also increased the secretion of IL‐6 from MΦ. However, when the IL‐33 was preincubated with ST2 prior to its addition to the MΦ cultures, the secretion of IL‐6 was attenuated. These data suggest that, though IL‐33 induced the Th2‐type immune responses and infiltration of M2 type MΦ into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.     

A two-step human culture system replicates intestinal monocyte maturation cascade: Conversion of tissue-like inflammatory monocytes into macrophages

Monocyte maturation program into macrophages (MΦ) is well defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicated the human monocyte “waterfall” process in vitro by first, generating tissue-like inflammatory monocytes, which were then shifted toward MΦ. Purified CD14⁺CD16⁻ monocytes, cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ and IL23, differentiated into CD14⁺CD163⁻ cells that displayed a monocyte-like morphology. In vitro generated inflammatory CD14⁺CD163⁻ (inflammatory monocyte-like cells) cells promoted IL-1β-dependent memory Th17 and Th17/Th1 responses, like the CD14⁺CD163⁻ mo-like cells that accumulate in inflamed colon of Crohn's disease patients. Next, in vitro generated inflammatory monocyte-like cells converted to functional CD163⁺ MΦ following exposure to TGF-β and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163⁺ MΦ and MΦ detected in various inflamed nonlymphoid and lymphoid diseased tissues. Our findings propose a two-step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulation of this process might open therapeutic avenues for chronic inflammatory disorders.     

Spleen-derived macrophages are readily polarized into classically activated (M1) or alternatively activated (M2) states

Bone marrow derived macrophages (BM-MΦ) that differentiate from precursor cells can be polarized into classically activated pro-inflammatory (M1) or alternatively activated (M2) states depending upon the cytokine microenvironment. We questioned whether tissue MΦ, such as spleen-derived MΦ (Sp-MΦ), have the ability to differentiate into M1 or M2 cells. We show in response to activation with IFN-gamma (IFN-γ) and lipopolysaccharide (LPS), that the Sp-MΦ readily acquired an M1 status indicated by up-regulation of iNOS mRNA, nitric oxide (NO) production, and the co-stimulatory molecule CD86. Conversely, Sp-MΦ exposed to IL-4 exhibited increased levels of mannose receptor (CD 206), arginase-1 (Arg)-1 mRNA expression, and significant urea production typical of M2 cells. At this stage of differentiation, the M2 Sp-MΦ were more efficient at phagocytosis of cell-associated antigens than their M1 counterparts. This polarization was not indefinite as the cells could revert back to their original state upon the removal of stimuli and exhibited flexibility to convert from M2 to M1. Remarkably, both M1 and M2 Sp-MΦ induced more CD4 expression on their cells surface after stimulation. We also demonstrate that adherent macrophages cultured for a short term in IL-4 enhances ARG-1 and YM-1 mRNA along with increasing urea producing capacity: traits indicative of an M2 phenotype. Moreover, in response to in vivo virus infection, the adherent macrophages obtained from spleens rapidly express iNOS. These results provide new evidence for the polarization capabilities of Sp-MΦ when exposed to pro-inflammatory or anti-inflammatory cytokines.     

A role for IL‐18 in protective immunity against Mycobacterium tuberculosis

Tuberculosis remains the most hazardous bacterial infection worldwide. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular pathogen of resting MΦ. IFN‐γ secreted by natural killer, CD4 Th 1 and CD8 T cells upon instruction by IL‐12 and ‐18 activates MΦ to restrict mycobacterial growth. Production of both cytokines is induced by TLR signalling in DC and MΦ. Mice deficient for the TLR adaptor, MyD88, are highly susceptible to M. tuberculosis infection. Shared usage of MyD88 by signalling cascades for TLR and receptors for IL‐1 and IL‐18 prompted us to revisit the role of IL‐18 during experimental infection with M. tuberculosis. We show that mice deficient for IL‐18 and MyD88 but not for IL‐18 receptor promptly succumbed to M. tuberculosis infection in contrast to WT or TLR‐2/‐4 double KO mice indicating that lack of IL‐18 contributes to the high susceptibility of MyD88 KO mice to M. tuberculosis. Without IL‐18, the protective Th1 response was decreased and hence, mycobacterial propagation was favoured. Neutrophil‐driven lung immunopathology concomitant with unrestrained growth of tubercle bacilli are most likely responsible for the premature death of IL‐18 KO mice. Thus, IL‐18 plays a decisive role in protective immunity against tuberculosis.     

Natural killer cell-mediated response to human cytomegalovirus-infected macrophages is modulated by their functional polarization

MΦ comprise a heterogeneous population of cells, which contribute to host defense and maintenance of immune homeostasis. MΦ may be infected by human cytomegalovirus (HCMV), which has evolved different strategies to subvert the immune response. In the present study, we comparatively analyzed the natural killer (NK) cell response against HCMV (TB40E)-infected proinflammatory (M1) and antinflammatory (M2) MΦ, derived from autologous monocytes, cultured in the presence of GM-CSF and M-CSF, respectively. M1 MΦ were more resistant to infection and secreted IL-6, TNF-α, IFN-α, and IL-12; by contrast, in HCMV-infected M2 MΦ, proinflammatory cytokines, IL-10, and IFN-α production were limited and IL-12 was undetectable. NK cell degranulation was triggered by interaction with HCMV-infected M1 and M2 MΦ at 48 h postinfection. The response was partially inhibited by specific anti-NKp46, anti-DNAM-1, and anti-2B4 mAb, thus supporting a dominant role of these activating receptors. By contrast, only HCMV-infected M1 MΦ efficiently promoted NK cell-mediated IFN-γ secretion, an effect partially related to IL-12 production. These observations reveal differences in the NK cell response triggered by distinct, HCMV-infected, monocyte-derived cell types, which may be relevant in the immunopathology of this viral infection.     

Engulfment of Hb‐activated platelets differentiates monocytes into pro‐inflammatory macrophages in PNH patients

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)‐activated platelets, classical monocytes differentiated into pro‐inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb‐activated platelets, monocytes differentiate into M1‐macrophages under M1‐polarization stimulus (GM‐CSF, IFN‐γ + LPS). When grown under M2‐polarization stimulus (M‐CSF, IL‐4 + IL13), the cells exhibited an M1‐like phenotype, secreted elevated levels of pro‐inflammatory cytokines including TNF‐α and IL‐1β, and displayed loss of the secretion of cytokine such as IL‐10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho‐STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte–platelet aggregates in circulation and accumulation of CD80^high macrophages in thioglycollate‐induced inflamed peritoneum. The spleen of the mice was also populated by CD80^high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb‐activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.     

F4/80, a monoclonal antibody directed specifically against the mouse macrophage

A hybridoma clone which secretes a macrophage (MΦ)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal MΦ with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG_2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the MΦ and, at least in part, exposed on the cell surface.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi

Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.     

Distinct expression of interleukin (IL)‐36α, β and γ, their antagonist IL‐36Ra and IL‐38 in psoriasis,rheumatoid arthritis and Crohn's disease

Interleukin (IL)‐36α, IL‐36β and IL‐36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL‐36Ra or IL‐38, another potential IL‐36 inhibitor, limit uncontrolled inflammation. The expression and role of IL‐36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod‐induced mouse skin inflammation and in human psoriasis, expression of IL‐36α, γ and IL‐36Ra, but not IL‐36β and IL‐38 mRNA, was induced and correlated with IL‐1β and T helper type 17 (Th17) cytokines (IL‐17A, IL‐22, IL‐23, CCL20). In mice with collagen‐induced arthritis and in the synovium of patients with RA, IL‐36α, β, γ, IL‐36Ra and IL‐38 were all elevated and correlated with IL‐1β, CCL3, CCL4 and macrophage colony‐stimulating factor (M‐CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium‐induced colitis and in patients with CD, only IL‐36α, γ and IL‐38 were induced at relatively low levels and correlated with IL‐1β and IL‐17A. We suggest that only a minor subgroup of patients with RA (17–29%) or CD (25%) had an elevated IL‐36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL‐36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68⁺ macrophages, dendritic/Langerhans cells and CD79α⁺ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL‐36β and IL‐36Ra were produced constitutively, but IL‐36α, γ and IL‐38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL‐36 agonists/antagonists ratio.     

The purinergic P2×7 receptor is expressed on monocytes in Behçet's disease and is modulated by TNF‐α

The P2×7 receptor (P2×7r) is expressed in innate immune cells (e.g. monocyte/macrophages), playing a key role in IL‐1β release. Since innate immune activation and IL‐1β release seem to be implicated in Behçet's disease (BD), a systemic immune‐inflammatory disorder of unknown origin, we hypothesized that P2×7r is involved in the pathogenesis of the disease. Monocytes were isolated from 18 BD patients and 17 healthy matched controls. In BD monocytes, an increased P2×7r expression and Ca²⁺ permeability induced by the selective P2×7r agonist 2′‐3′‐O‐(4‐benzoylbenzoyl)ATP (BzATP) was observed. Moreover, IL‐1β release from LPS‐primed monocytes stimulated with BzATP was markedly higher in BD patients than in controls. TNF‐α‐incubated monocytes from healthy subjects almost reproduced the findings observed in BD patients, as demonstrated by the increase in P2×7r expression and BzATP‐induced Ca²⁺ intake. Our results provide evidence that in BD monocytes both the expression and function of the P2×7r are increased compared with healthy controls, as the possible result, at least in part, of a positive modulating effect of TNF‐α on the receptor. These data indicate P2×7r as a new potential therapeutic target for the control of BD, further supporting the rationale for the use of anti‐TNF‐α drugs in the treatment of the disease.     

T‐helper 1 and T‐helper 2 adjuvants induce distinct differences in the magnitude,quality and kinetics of the early inflammatory response at the site of injection

Vaccine adjuvants activate the innate immune system and thus influence subsequent adaptive T‐cell responses. However, little is known about the initial immune mechanisms preceding the adjuvant‐induced differentiation of T‐helper (Th) cells. The effect of a T‐helper 1 (Th1) adjuvant, dimethyldioctadecylammonium liposomes with monophosphoryl lipid‐A (DDA/MPL), and a T‐helper 2 adjuvant, aluminium hydroxide [Al(OH)₃], on early, innate chemotactic signals and inflammatory cell influx at the site of injection was therefore investigated. Injection of the adjuvants into the peritoneal cavity of mice demonstrated distinct differences in the magnitude, quality and kinetics of the response. The inflammatory response to DDA/MPL was prominent, inducing high local levels of pro‐inflammatory cytokines, chemokines and a pronounced inflammatory exudate consisting of neutrophils, monocytes/macrophages and activated natural killer cells. This was in contrast to the response induced by Al(OH)₃, which, although sharing some of the early chemokine signals, was more moderate and consisted almost exclusively of neutrophils and eosinophils. Notably, Al(OH)₃ specifically induced the release of a significant amount of interleukin (IL)‐5, whereas DDA/MPL induced high amounts of tumour necrosis factor‐α (TNF‐α), IL‐1α and IL‐6. Finally, a microarray analysis confirmed that the effect of DDA/MPL was broader with more than five times as many genes being specifically up‐regulated after injection of DDA/MPL compared with Al(OH)₃. Thus, the adjuvants induced qualitatively distinct local inflammatory signals early after injection.     

Histones trigger sterile inflammation by activating the NLRP3 inflammasome

Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll‐like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)–1β and IL‐18. Here, we report that histones released from necrotic cells induce IL‐1β secretion in an NLRP3–ASC‐caspase‐1‐dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone‐induced IL‐1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone‐neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell‐derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL‐1β secretion via oxidative stress.     

Macrophages participate in IL-17-mediated inflammation

The involvement of macrophages (MΦs) in Th17-cell responses is still poorly understood. While neutrophils are thought to be the predominant effector of Th17-cell responses, IL-17 is also known to induce myelotropic chemokines and growth factors. Other T-cell-derived cytokines induce non-classical functions, suggesting that IL-17 sigxnaling may similarly elicit unique MΦ functions. Here, we characterized the expression of subunits of the IL-17 receptor on primary murine MΦs from different anatomical compartments. The greatest expression of IL-17 receptors was observed on mucosal Ly6C(hi) "inflammatory" MΦs. We further observed upregulation of IL-17 receptors in vitro on bone marrow-derived macrophages (BMMΦs) in response to peptidoglycan or CpG oligonucleotide stimuli, and in vivo, upon CFA administration. Macrophages expressing IL-17 receptors were observed infiltrating the hearts of mice with myocarditis, and genetic ablation of IL-17RA altered MΦ recruitment. Treating primary MΦs from a wide variety of different anatomic sources (as well as cell lines) with IL-17A induced the production of unique profiles of cytokines and chemokines, including GM-CSF, IL-3, IL-9, CCL4/MIP-1β and CCL5/RANTES. IL-17A also induced production of IL-12p70; IL-17-signaling-deficient MΦs elicited diminished IFN-γ production by responding DO11.10 CD4(+) T cells when used as APCs. These data indicate that MΦs from different anatomic locations direct IL-17-mediated responses.     

CD137 ligand signaling induces human monocyte to dendritic cell differentiation

The ligand for CD137 (4‐1BB) is expressed on peripheral human monocytes and delivers a potent activating signal via reverse signaling. Here we show that treatment of monocytes with a recombinant CD137 protein that induces reverse signaling through CD137 ligand reduces typical macrophage characteristics such as phagocytosis, oxidative burst and CD14 expression; however, typical DC characteristics including endocytosis, costimulatory molecule expression and the ability to stimulate proliferation of naïve T cells are induced. CD137‐generated DC do not express DC‐SIGN, CD1a or IL‐12 and secrete less IL‐10 than classical DC. CD137‐generated DC are mature, and addition of LPS+IFN‐γ does not enhance their T‐cell‐stimulatory capacity. This indicates that CD137 as a sole factor is sufficient to induce development to mature DC, making stimulation of CD137 ligand the most simple protocol to generate mature DC. CD137‐generated DC are more potent in inducing T‐cell proliferation than classical DC. They inhibit development of Treg cells but induce T‐cell expression of perforin, IFN‐γ, IL‐13 and IL‐17. These data demonstrate that CD137 as a single factor is sufficient to induce differentiation of peripheral monocytes to mature inflammatory DC that have a more potent T‐cell‐stimulatory capacity than classical DC.