NLRP3 inflammasome mediates interleukin‐1β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

Acinetobacter baumannii is a multi‐drug resistant, Gram‐negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase‐1, and their activation is required for maturation of interleukin‐1β (IL‐1β). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase‐1, but not NLRC4, are required for A. baumannii‐induced production of IL‐1β in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K⁺ efflux, reactive oxygen species production and release of cathepsins, are involved in IL‐1β production in macrophages in response to A. baumannii. Interleukin‐1β production in bronchoalveolar lavage (BAL) fluid was impaired in NLRP3‐deficient and caspase‐1/11‐deficient mice infected with A. baumannii, compared with that in wild‐type (WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3‐deficient or caspase‐1/11‐deficient mice. The severity of lung pathology was reduced in NLRP3‐ deficient, caspase‐1/11‐ deficient and IL‐1‐receptor‐deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL‐1β production and lung pathology.     

Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL‐1β secretion in murine macrophages

NLRP3 inflammasome is a protein complex crucial to caspase‐1 activation and IL‐1β and IL‐18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus‐induced IL‐1β secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7‐dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL‐1β secretion in infected murine macrophages. Additionally, our results suggest that B. abortus‐induced IL‐1β secretion depends on a P2X7‐independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.     

IL‐37 Expression is Upregulated in Patients with Tuberculosis and Induces Macrophages Towards an M2‐like Phenotype

Interleukin‐37 (IL‐37), a member of the IL‐1 family, primarily functions as an anti‐inflammatory cytokine, reducing inflammation and suppressing the immune response. However, the expression and role of IL‐37 in tuberculosis (TB) remains unknown. We aimed to measure serum levels of IL‐37 and several important cytokines in 25 patients with active TB and to analyse their association with disease activity. We found that IL‐37 levels decreased in patients with TB and recovered after treatment. IL‐37 levels negatively correlated with the serum concentration of IFN‐γ and IL‐12 but positively correlated with IL‐10 and TGF‐β levels. After IL‐37, secretion was blocked in peripheral blood mononuclear cells from active patients with TB, IFN‐γ and IL‐10 production was significantly upregulated; this was not observed in healthy donors or patients after treatment. IL‐37 knockdown significantly enhanced the phagocytic activity of THP1‐derived macrophages towards Mycobacterium tuberculosis (M. tb). M1/M2 polarization‐associated markers were detected simultaneously, and IL‐37 induced a phenotypic shift in THP1‐derived macrophages towards a high CD206⁺ and low CD86⁺ macrophage subtype. Furthermore, this phenotypic shift was accompanied by upregulated mRNA levels of arginase 1, TGF‐β and IL‐10, which are characteristic hallmarks of M2 macrophages. In conclusion, our results suggest that increased levels of IL‐37 in patients with TB are associated with IFN‐γ, IL‐12, IL‐10 and TGF‐β levels and that IL‐37 plays a pathological role in TB infection by inhibiting the production of pro‐inflammatory cytokines and inducing macrophages towards an M2‐like phenotype. Thus, IL‐37 may be a novel research target to understand the pathogenesis of TB infection.     

Cytotoxins of the human pathogen Aeromonas hydrophila trigger,via the NLRP3 inflammasome,caspase‐1 activation in macrophages

Aeromonas hydrophila is a Gram‐negative pathogen that causes serious infectious disease in humans. A. hydrophila induces apoptosis in infected macrophages, but the host proinflammatory responses triggered by macrophage death are largely unknown. Here, we demonstrate that the infection of mouse macrophages with A. hydrophila triggers the activation of caspase‐1 and release of IL‐1β. Caspase‐1 activation was abrogated in macrophages deficient in Nod‐like receptor family, pyrin domain containing 3 (NLRP3) and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), but not NLR family, CARD domain containing 4 (NLRC4). The activation of the NLRP3 inflammasome was mediated by three cytotoxins (aerolysin, hemolysin and multifunctional repeat‐in‐toxin) produced by A. hydrophila. Our results indicated that the NLRP3 inflammasome senses A. hydrophila infection through the action of bacterial cytotoxins.     

Impaired NLRP3 inflammasome activity during fetal development regulates IL‐1β production in human monocytes

Interleukin‐1β (IL‐1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll‐like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16^pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro‐IL‐1β precursor protein upon LPS stimulation. In contrast, high levels of pro‐IL‐1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL‐1β. The lack of secreted IL‐1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase‐1 activity before 29 weeks of gestation, whereas expression of the apoptosis‐associated speck‐like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP‐induced caspase‐1 activity in cord blood monocytes. Lastly, secretion of IL‐1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL‐1β responses early in gestation, in part due to a downregulation of TLR‐mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.     

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.     

Commensal microflora and interferon‐γ promote steady‐state interleukin‐7 production in vivo

IL‐7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL‐7 production. To study Il7 gene regulation in vivo, we generated a novel IL‐7‐reporter mouse, which allows the non‐invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL‐7‐producing cells. With these IL‐7‐reporter mice, we identify thymus, skin and intestine as major sources of IL‐7 in vivo. Importantly, we show that IFN‐γ and the commensal microflora promote steady‐state IL‐7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN‐γ signaling in intestinal epithelial cells strongly reduces their IFN‐γ‐driven IL‐7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN‐γ production by lymphocytes, which in turn promotes epithelial cell IL‐7 production and the survival of IL‐7‐dependent lymphocytes.     

Negative regulation of Nod‐like receptor protein 3 inflammasome activation by T cell Ig mucin‐3 protects against peritonitis

The Nod‐like receptor protein 3 (NLRP3) inflammasome plays roles in host defence against invading pathogens and in the development of autoimmune damage. Strict regulation of these responses is important to avoid detrimental effects. Here, we demonstrate that T cell Ig mucin‐3 (Tim‐3), an immune checkpoint inhibitor, inhibits NLRP3 inflammasome activation by damping basal and lipopolysaccharide‐induced nuclear factor‐κB‐mediated up‐regulation of NLRP3 and interleukin‐1β during the priming step and basal and ATP/lipopolysaccharide‐induced ATP production, K⁺ efflux, and reactive oxygen species production during the activation step. Residues Y256/Y263 in the C‐terminal region of Tim‐3 are required for these inhibitory effects on the NLRP3 inflammasome. In mice with alum‐induced peritonitis, blockade of Tim‐3 exacerbates peritonitis by overcoming the inhibitory effect of Tim‐3 on NLRP3 inflammasome activation, while transgenic expression of Tim‐3 attenuates inflammation by inhibiting NLRP3 inflammasome activation. Our results show that Tim‐3 is a critical negative regulator of NLRP3 inflammasome and provides a potential target for intervention of diseases with uncontrolled inflammasome activation.     

RGS16 Restricts the Pro‐Inflammatory Response of Monocytes

Immune cells express powerful and harmful effectors that require tight regulation. Heterotrimeric G proteins are critical mediators in translating extracellular signals into cell responses, which need a fine‐tuned regulation for the control of cell activation. Regulator of G‐protein signalling 16 (RGS16) has been identified as a key factor of G protein‐mediated activation in lymphocytes, modulating inflammatory and survival responses of various cell types. However, data about the expression of this regulatory protein in monocytes are scarce, and it has remained unclear whether activation and migration of these cells are regulated by RGS16. In this study, the impact of RGS16 on the production of inflammatory cytokines by activated human monocytes was investigated in vitro using the human promonocytic cell line THP‐1 as a model. Gain and loss of function experiments showed that RGS16 overexpression reduces the expression of pro‐inflammatory cytokines IL‐1β, IL‐6, IL‐8 and TNFα, while RGS16 knockdown by RNAi upregulates IL‐1β, IL‐6 and TNFα but not IL‐8. RGS16 knockdown was also shown to enhance Pam3‐mediated induction of the anti‐inflammatory cytokine IL‐10. Our results indicate that RGS16 restricts the activation‐induced pro‐inflammatory profile in myeloid cells.     

High‐density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation

Interleukin‐1β (IL‐1β), a potent pro‐inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL‐1β is controlled by a multi‐protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High‐density lipoprotein (HDL) has consistently been shown to be anti‐atherogenic and to have anti‐inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL‐1β secretion in response to cholesterol crystals in THP‐1 cells and in human‐monocyte‐derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro‐IL‐1β and NLRP3, and reducing caspase‐1 activation. In summary, we have described a novel anti‐inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.     

Innate IFN‐γ Production by Subsets of Natural Killer Cells,Natural Killer T Cells and γδ T Cells in Response to Dying Bacterial‐Infected Macrophages

Interferon‐γ (IFN‐γ) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN‐γ is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)‐12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL‐12p70 unless these cells are primed by IFN‐γ. It remains, therefore, unsettled how initial IFN‐γ is produced. In a previous study, we reported a novel IFN‐γ production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone‐marrow‐derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN‐γ production pathway. We also unequivocally demonstrated by using BMM cells from IL‐12‐deficient mice that the bacterial‐infected macrophage cell death‐mediated IFN‐γ production can occur without IL‐12 although the magnitude of the response is much smaller than that in the presence of IL‐12. The enhancing effect of IL‐12 on this response proved to be attributable to the negligible amounts (0.5～1.5 pg/ml) of IL‐12p70 but not to the large amounts of IL‐12p40 that were both secreted by SA‐ and LM‐infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN‐γ production at an early stage of bacterial infection.     

Interferon‐γ constrains cytokine production of group 2 innate lymphoid cells

Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin‐5 (IL‐5), which supports eosinophil responses in various tissues; they also produce IL‐13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL‐33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon‐γ (IFN‐γ). Interferon‐γ severely inhibited IL‐5 and IL‐13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α‐galactosylceramide (α‐GalCer) to induce NKT cells to produce IL‐33 and IFN‐γ. Intraperitoneal injection of α‐GalCer in mice induced NKT cell activation resulting in IL‐5 and IL‐13 production by ILC2s. Administration of anti‐IFN‐γ together with α‐GalCer significantly enhanced the production of IL‐5 and IL‐13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL‐33 in Il33^?/? mice pre‐treated with α‐GalCer. Hence, IFN‐γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.     

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.     

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.