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.     

Differential susceptibility to lethal endotoxaemia in mice deficient in IL-1α, IL-1β or IL-1 receptor type I

Joosten LAB, van de Veerdonk F, Vonk AG, Boerman OC, Keuter M, Fantuzzi G, Verschueren I, van der Poll T, Dinarello CA, Kullberg BJ, Van der Meer JWM, Netea MG. Differential susceptibility to lethal endotoxaemia in mice deficient in IL‐1α, IL‐1β or IL‐1 receptor type I. APMIS 2010; 118: 1000–7. The role of intereukin‐1 (IL‐1) in mortality caused by endotoxaemia remains controversial. While IL‐1 receptor antagonist (IL‐1Ra) protects mice from lethal endotoxaemia, mice deficient in IL‐1β (IL‐1β^{? /?}) display normal susceptibility to lipopolysaccharide (LPS). The aim of this study was to identify the source of these discrepancies. Mice deficient in IL‐1α, IL‐1β or IL‐1R type I were injected intraperitoneally with Escherichia coli or Salmonella typhimurium LPS. Survival of the mice was examined and compared with C57/Bl6 wild‐type mice. In addition, serum cytokine concentrations were determined after LPS challenge and in vitro cytokine production by peritoneal macrophages was analysed. Clearance of radioactive IL‐1α was examined in IL‐1α^?/? and wild‐type mice. IL‐1β^?/? mice were normally susceptible to endotoxaemia and cytokine production did not differ from that in control mice. Surprisingly, LPS mortality in IL‐1α^?/? mice was significantly greater than that in control mice, accompanied by higher interferon‐γ release. These effects were mediated by a distorted homeostasis of IL‐1RI receptors, as shown by a strongly delayed clearance of IL‐1α. In contrast to the IL‐1α^?/? and IL‐1β^?/? mice, IL‐1RI^?/? mice were completely resistant to high doses of LPS. In conclusion, IL‐1RI‐mediated signals are crucial in mediating mortality occurring as a result of lethal endotoxaemia. Investigation of IL‐1‐mediated pathways in IL‐1 knock‐out mice is complicated by a distorted homeostasis of IL‐1Rs.     

IL‐1β activation in response to Staphylococcus aureus lung infection requires inflammasome‐dependent and independent mechanisms

Maintaining balanced levels of IL‐1β is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1^?/? mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1^?/? mice with S. aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL‐1β. Ifnlr1^?/? mice treated with recombinant IL‐1β displayed increased bacterial burdens in the airway and lung. IL‐1β levels in neutrophils from Ifnlr1^?/? infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase‐1 had reduced IL‐1β levels 4 h after infection, due to reductions or absence of active caspase‐1 respectively, but levels at 24 h were comparable to WT infected mice. Ifnlr1^?/? infected mice had decreases in both active caspase‐1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL‐1β processing. By inhibiting neutrophil elastase, we were able to decrease IL‐1β levels by 39% in Nlrp3^?/? infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL‐1β processing, via inflammasome‐dependent and ‐independent mechanisms.     

IL‐13Rα1 is a surface marker for M2 macrophages influencing their differentiation and function

In this study, we examined the role IL‐13 receptor alpha 1 (IL‐13Rα1) plays in macrophage differentiation and function. The findings indicate that IL‐13Rα1 is expressed on the M2 but not on the M1 subset of macrophages and specifically heterodimerizes with the IL‐4Rα chain to form a type II receptor, which controls the differentiation and function of these cells. Indeed, BM cells from IL‐13Rα1^+/+ and IL‐13Rα1^?/? mice yield equivalent numbers of macrophages when cultured under M2 polarizing conditions. However, IL‐13Rα1^?/? BM cells yield a much higher number of macrophages than IL‐13Rα1^+/+ BM cells when the differentiation is carried out under M1‐polarizing conditions. Further analyses indicated that macrophages that express IL‐13Rα1 also display surface markers associated with an M2 phenotype. In addition, the IL‐13Rα1⁺ macrophages were highly efficient in phagocytizing zymosan bioparticles both in vitro and in vivo, and supported differentiation of naïve T cells to a Th2 phenotype. Finally, when stimulated by IL‐13, a cytokine that uses the heteroreceptor, the cells were able to phosphorylate STAT6 efficiently. These previously unrecognized findings indicate that IL‐13Rα1 serves as a marker for M2 macrophages and the resulting heteroreceptor influences both their differentiation and function.     

IL-10 contributes to the suppressive function of tumour-associated myeloid cells and enhances myeloid cell accumulation in tumours

Studies have revealed that tumour‐associated myeloid cells (TAMC) are one of the major sources of IL‐10 in tumour‐bearing mice. However, the significance of TAMC‐derived IL‐10 in tumour immunity is poorly understood. Here, we show that IL‐10 blockade or IL‐10 deficiency reduces the capacity of TAMC in suppressing the proliferation of P1A‐specific CD8 T cells. In the spleen, IL‐10‐deficient and wild‐type (WT) mice bearing large tumour burdens have similar TAMC populations. The tumours from IL‐10‐deficient mice, however, have reduced numbers of TAMC compared with tumours from their WT counterparts. IL‐10^?/?RAG‐2^?/? mice also had reduced numbers of TAMC compared with tumours from IL‐10^+/+RAG‐2^?/? mice; therefore, the reduction in TAMC in IL‐10‐deficient tumours was not because of adaptive immune response in tumours. Adoptively transferred tumour antigen–specific CD8 T cells expanded more efficiently within tumours in IL‐10^?/?RAG‐2^?/? mice than in tumours from IL‐10^+/+RAG‐2^?/? mice. Cytotoxic T lymphocyte adoptive transfer therapy prevented tumour evasion in IL‐10^?/?RAG‐2^?/? mice more efficiently than in IL‐10^+/+RAG‐2^?/? mice. Thus, IL‐10 enhances the accumulation of myeloid cells in tumours, and TAMC‐derived IL‐10 suppresses the activation and expansion of tumour antigen–specific T cells.     

SQSTM1/p62/A170 regulates the severity of Legionella pneumophila pneumonia by modulating inflammasome activity

Sequestosome1/A170/p62 (SQSTM1) is a scaffold multifunctional protein involved in several cellular events, such as signal transduction, cell survival, cell death, and inflammation. SQSTM1 expression by macrophages is induced in response to environmental stresses; however, its role in macrophage‐mediated host responses to environmental stimuli, such as infectious pathogens, remains unclear. In this study, we investigated the role of SQSTM1 in host responses to Legionella pneumophila, an intra‐cellular pathogen that infects macrophages, in both an SQSTM1‐deficient (SQSTM1^?/?) mouse model and macrophages from these mice. Compared with wild‐type (WT) macrophages, the production and secretion of the proinflammatory cytokine IL‐1β was significantly enhanced in SQSTM1^?/? macrophages after infection with L. pneumophila. Inflammasome activity, indicated by the level of IL‐18 and caspase‐1 activity, was also elevated in SQSTM1^?/? macrophages after infection with L. pneumophila. SQSTM1 may interact with nucleotide‐binding oligomerization domain‐like receptor family, caspase recruitment domain‐containing 4 and nucleotide‐binding oligomerization domain like receptor family, pyrin domain containing 3 proteins to inhibit their self‐dimerization. Acute pulmonary inflammation induced by L. pneumophila and silica was enhanced in SQSTM1^?/? mice with an increase in IL‐1β levels in the bronchoalveolar lavage fluids. These findings suggest that SQSTM1 is a negative regulator of acute pulmonary inflammation, possibly by regulating inflammasome activity and subsequent proinflammatory cytokine production.     

Protein tyrosine phosphatase PTPN22 regulates IL‐1β dependent Th17 responses by modulating dectin‐1 signaling in mice

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22^?/? mutant mice, were pulsed with OVA_323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22^?/? BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22^?/? BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22^R619W (the mouse orthologue of human PTPN22^R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22^R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.     

Lipocalin‐2 ensures host defense against Salmonella Typhimurium by controlling macrophage iron homeostasis and immune response

Lipocalin‐2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron‐laden bacterial siderophores, chemo‐attracts neutrophils and has immunomodulatory and apoptosis‐regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella‐infected macrophages, which reduces cellular iron content and enhances the generation of pro‐inflammatory cytokines. Lcn2 represses IL‐10 production while augmenting Nos2, TNF‐α, and IL‐6 expression. Lcn2^?/? macrophages have elevated IL‐10 levels as a consequence of increased iron content. The crucial role of Lcn‐2/IL‐10 interactions was further demonstrated by the greater ability of Lcn2^?/? IL‐10^?/? macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2^?/? counterparts. Overexpression of the iron exporter ferroportin‐1 in Lcn2^?/? macrophages represses IL‐10 and restores TNF‐α and IL‐6 production to the levels found in wild‐type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL‐10 production, and that both functions are linked to its ability to shuttle iron from macrophages.     

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.     

Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce cytokines, including tumor necrosis factor-α and interleukins (ILs), in the small intestine via a Toll-like receptor 4 (TLR4)-dependent pathway, leading to intestinal ulceration. Activation of the inflammasome promotes pro-caspase-1 cleavage, leading to pro-IL-1β maturation. We examined the role of NLRP3 inflammasome in NSAID-induced enteropathy. Small intestinal damage developed 3 h after indomethacin administration, accompanied by increases in IL-1β and NLRP3 mRNA expression and mature caspase-1 and IL-1β levels. In vivo blocking of IL-1β using neutralizing antibodies attenuated indomethacin-induced damage, whereas exogenous IL-1β aggravated it. NLRP3^−/− and caspase-1^−/− mice exhibited resistance to the damage with reduction of mature IL-1β production. This resistance was abolished by exogenous IL-1β. TLR4 deficiency prevented intestinal damage and inhibited upregulation of NLRP3 and IL-1β mRNAs and maturation of pro-caspase-1 and pro-IL-1β, whereas TLR4 activation by its agonists exerted opposite effects. Apyrase, an adenosine triphosphate (ATP) scavenger, or Brilliant Blue G, a purinergic P2X₇ receptor antagonist, inhibited the damage as well as caspase-1 activation and IL-1β processing, despite there being sufficient amounts of pro-IL-1β and NLRP3. These results suggest that NLRP3 inflammasome-derived IL-1β plays a crucial role in NSAID-induced enteropathy and that both TLR4- and P2X₇-dependent pathways are required for NLRP3 inflammasome activation.     

Immunisation route‐dependent expression of IL‐4/IL‐13 can modulate HIV‐specific CD8+ CTL avidity

All HIV‐1 ‘systemic vaccine trials’ in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8⁺ T cells generated higher levels of IL‐4/IL‐13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL‐4^?/? and IL‐13^?/? KO mice have shown that the capacity to express IFN‐γ, IL‐4 and/or IL‐13 by K^dGag_197–205‐specific CTL differed between these groups and was inversely correlated with CTL avidity (IL‐13^?/?>IL‐4^?/?>BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL‐13^?/? and wild type mice. When IL‐13 was reconstituted in IL‐13^?/? splenocytes in vitro, their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL‐13 can greatly enhance CTL avidity. In contrast, extracellular IL‐4 appears to be important in maintaining long‐term Th1/Th2 balance in CTL, even though expression of IL‐4 by CTL markedly reduced avidity. STAT6^?/? mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K^dGag_197–205‐specific CTL was also regulated by IL‐4/IL‐13.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

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.     

IL‐18 associates to microvesicles shed from human macrophages by a LPS/TLR‐4 independent mechanism in response to P2X receptor stimulation

Extracellular ATP, released upon microbial infection, cell damage, or inflammation, acts as an alert signal toward immune cells by activating P2 receptors. The nucleotide causes microvesicle (MV) shedding from immune and nonimmune cells. Here, we show that IL‐18 associates with MVs shed by human ex vivo macrophages upon P2X receptor stimulation. MV shedding was potently induced by ATP and by the P2X7 agonist 3′‐benzoylbenzoyl adenosine 5′‐triphosphate, while it was greatly reduced by P2X irreversible inhibitor‐oxidized ATP and by the specific P2X7 inhibitors KN‐62, A‐740003, and A‐438079. Peculiarly, the P2X7 subtype was highly present in the MVs, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca²⁺ ionophore A23187 mimicked the effect of 3′‐benzoylbenzoyl adenosine 5′‐triphosphate suggesting that an intracellular Ca²⁺ increase was sufficient to evoke MV shedding. Caspase inhibitors Ac‐YVAD‐CMK or Z‐YVAD‐CMK did not block the cleavage of MV‐associated pro‐IL‐18. Pro‐IL‐18 formation in macrophages did not require pretreatment of cells with LPS, as the procytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS‐binding antibiotic polymyxin B nor with the TLR‐4 intracellular inhibitor CLI‐095. These data reveal a nucleotide‐based mechanism responsible for the shedding of MV to which IL‐18 is associated.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.     

Card9‐dependent IL‐1β regulates IL‐22 production from group 3 innate lymphoid cells and promotes colitis‐associated cancer

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell‐specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL‐1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis‐associated cancer (CAC) model. Card9^?/? mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL‐1β generation and defective IL‐1β controlled IL‐22 production from group 3 innate lymphoid cells. Consistent with the key role of immune‐derived IL‐22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9^?/? mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9‐controlled, ILC3‐mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9‐mediated innate immunity in inflammation‐associated carcinogenesis.     

Expression of activation‐induced cytidine deaminase enhances the clearance of pneumococcal pneumonia: evidence of a subpopulation of protective anti‐pneumococcal B1a cells

We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation‐induced cytidine deaminase (AID^?/?), or invariant natural killer T (iNKT) cells (Jα18^?/?), or interleukin‐13 (IL‐13^?/?) had impaired early clearance of pneumococci in the lung, compared with wild‐type mice. In contrast, AID^?/? mice adoptively transferred with AID^+/+ B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity‐like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen‐specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti‐pneumococcal B1a cell initiating response, probably through early production of IL‐13, given that IL‐13^?/? mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID‐dependent subset.     

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.