IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

IL‐1‐dependent,IL‐1R1‐independent resistance to gastrointestinal nematodes

IL‐1 null mice are unable to expel the intestinal nematode Trichuris muris; whereas WT littermates exhibit sterile immunity. Intriguingly the essential signalling components IL‐1R1 and IL‐1R accessory protein (AcP) are dispensable for expulsion of this parasite. IL‐1 is thus critical for CD4⁺ Th2‐mediated immunity to T. muris; however, this action is independent of the established IL‐1 signalling receptor. We also present data demonstrating that both IL‐1α and IL‐1β induce measurable effects on T. muris primed cells isolated from IL‐1R1 or IL‐1R AcP null mice. MLN cells from these mice restimulated with parasite antigen proliferated at a greater rate and produced more cytokines in response to exogenous IL‐1. This ability to respond to IL‐1 was restricted to these parasite‐primed cells and importantly was not evident in cells from naïve gene null mice. These in vitro data are consistent with the observed ability of mice with compromised IL‐1 signalling to expel the parasite, bolstering the premise that an alternative IL‐1 signalling mechanism is accessible in the context of an intestinal helminth‐driven Th2 immune response.     

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.     

Nodal promotes the generation of M2‐like macrophages and downregulates the expression of IL‐12

Nodal, a member of the TGF‐β superfamily, is an embryonic morphogen that is upregulated in different types of tumors. Nodal increases the tumorigenesis by inducing angiogenesis and promoting metastasis. Importantly, Nodal inhibition suppresses the growth and invasion of tumor. Since tumor‐associated macrophages (TAMs) are the major infiltrating leukocytes in most cancers, we investigated whether Nodal is involved in the differentiation of TAMs. Our results revealed that Nodal inhibition in tumor microenvironment upregulated the production of IL‐12 in macrophages and reversed TAMs to classically activated macrophage phenotype. In contrast, treatment with recombinant Nodal (rNodal) decreased the expression of IL‐12 in murine macrophages. Furthermore, rNodal promoted macrophage polarization to an alternatively activated macrophage‐like/TAM phenotype and modulated its function. These results suggest that Nodal may play an important role in macrophage polarization and downregulation of IL‐12. The rescued antitumor function of TAMs via the inhibition of Nodal expression could be a new therapeutic strategy for cancer treatment.     

Inhibition of blood vessel formation by a chondrocyte-derived extracellular matrix

In this study, the chondrocyte-derived extracellular matrix (CECM) was evaluated for its activity to inhibit vessel invasion in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) and rabbit chondrocytes were plated on a bio-membrane made of CECM or human amniotic membrane (HAM). The adhesion, proliferation, and tube formation activity of HUVECs and chondrocytes were examined. The CECM and HAM powders were then mixed individually in Matrigel and injected subcutaneously into nude mice to examine vessel invasion in vivo after 1 week. Finally, a rabbit model of corneal neovascularization (NV) was induced by 3-point sutures in the upper cornea, and CECM and HAM membranes were implanted onto the corneal surface at day 5 after suture injury. The rabbits were sacrificed at 7 days after transplantation and the histopathological analysis was performed. The adhesion and proliferation of HUVECs were more efficient on the HAM than on the CECM membrane. However, chondrocytes on each membrane showed an opposite result being more efficient on the CECM membrane. The vessel invasion in vivo also occurred more deeply and intensively in Matrigel containing HAM than in the one containing CECM. In the rabbit NV model, CECM efficiently inhibited the neovessels formation and histological remodeling in the injured cornea. In summary, our findings suggest that CECM, an integral cartilage ECM composite, shows an inhibitory effect on vessel invasion both in vitro and in vivo, and could be a useful tool in a variety of biological and therapeutic applications including the prevention of neovascularization after cornea injury.     

IL‐18, but not IL‐15, contributes to the IL‐12‐dependent induction of NK‐cell effector functions by Leishmania infantum in vivo

Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK‐cell response in mice that required TLR9‐positive myeloid DC and IL‐12, but no IFN‐α/β. Here, we investigated whether IL‐15 or IL‐18 mediate the activity of IL‐12 or function as independent activators of NK cells. In contrast to earlier studies that described IL‐15 as crucial for NK‐cell priming in response to TLR ligands, the expression of IFN‐γ, FasL, perforin and granzyme B by NK cells in L. infantum‐infected mice was completely preserved in the absence of IL‐15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN‐γ secretion, cytotoxicity and FasL expression of NK cells from infected IL‐18^?/? mice were significantly reduced compared with controls, but, unlike IL‐12, IL‐18 was not essential for NK‐cell effector functions. Part of the NK‐cell‐stimulatory effect of IL‐12 was dependent on IL‐18. We conclude that IL‐15 is not functioning as a universal NK‐cell priming signal and that IL‐18 contributes to the NK‐cell response in visceral leishmaniasis. The cytokine requirements for NK‐cell activation appear to differ contingent upon the infectious pathogen.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

IL‐1β, IL‐18, and eicosanoids promote neutrophil recruitment to pore‐induced intracellular traps following pyroptosis

Inflammasomes activate caspase‐1, initiating a lytic form of programmed cell death termed pyroptosis, which is an important innate immune defense mechanism against intracellular infections. We recently demonstrated in a mouse infection model of pyroptosis that instead of releasing bacteria into the extracellular space, bacteria remain trapped within the pyroptotic cell corpse, termed the pore‐induced intracellular trap (PIT). This trapping mediates efferocytosis of the PIT and associated bacteria by neutrophils; bacteria are subsequently killed via neutrophil ROS. Using this pyroptosis model, we now show that the pro‐inflammatory cytokines IL‐1β and IL‐18 and inflammatory lipid mediators termed eicosanoids are required for effective clearance of bacteria downstream of pyroptosis. We further show that IL‐1β, IL‐18, and eicosanoids affect this in part by mediating neutrophil recruitment to the PIT. This is in addition to our prior findings that complement is also important to attract neutrophils. Thus, the PIT initiates a robust and coordinated innate immune response involving multiple mediators that attract neutrophils to efferocytose the PIT and its entrapped bacteria.     

Early onset wheeze associated with enhanced combined IL‐1β, IL‐6, and IL‐12/IL‐23p40 in LPS‐stimulated cord blood mononuclear cells

Background Neonates with a family history of atopy are at higher risk for developing wheezing in early life. Objective From a birth cohort of at risk infants (first‐degree family with atopic disease), we evaluated the influence of distinct intrinsic immunologic risk factors on wheezing disorders in the first 2 years of life. Methods Cord blood samples were collected from 195 eligible subjects of a birth cohort of 253 subjects. The subjects studied were those who developed wheezing (n=34) or eczema (n=29) in the first 2 years of life, and 65 healthy control infants. At the time of thawing the viability of the cells were median 70% (range 67.5%–72.5%). Cytokines from lipopolysaccharide (LPS)‐stimulated mononuclear cells were analysed using fluorescent‐activated cell sorting‐array and their profiles were evaluated using factor analysis. Results Infants with wheeze were significantly associated with enhanced combined LPS stimulated IL‐1β, IL‐6, and IL‐12/IL‐23p40 compared with healthy controls (P=0.003). This profile was also associated with the increased risk for wheeze at 2 years of age (OR=2.45; 95% CI=1.50–3.93, P=0.001). LPS‐stimulated cytokine IL‐8 was also significantly higher in the wheeze group compared with healthy controls and eczema (P=0.003). Intracellular staining showed that monocytes are main producers of IL‐6 and IL‐8 from cord blood mononuclear cells. Most of the subjects were non‐atopic with 3/34 (9%) wheeze and 9/29 (31%) eczema subjects sensitized to the common dietary or inhalant allergens. Conclusion and Clinical Relevance In infants at genetic risk of atopy, wheeze but not eczema in the first 2 years of life is associated with intrinsic hyperresponsive innate cytokine responses which might predispose infants to wheeze development. Distinct pre‐symptomatic hyperresponsive innate immune responses risk factors were found to be associated with early onset wheeze disorders, but not eczema. Cite this as: P. L. Quah, I‐C Kuo, C. H. Huang, L. P‐C Shek, B. W. Lee and K. Y. Chua, Clinical & Experimental Allergy, 2011 (41) 970–978.     

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis,selectively induces pro‐inflammatory cytokines and IL‐10 and is regulated by TNF‐α, IL‐10 and IFN‐γ

Leukocyte immunoglobulin‐like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane‐bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross‐linking of LILRA5 on monocytes induced production of pro‐inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common γ chain of the FcR and LILRA5 cross‐linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro‐inflammatory cytokines as well as IL‐10. LILRA5 mRNA and protein expression was tightly regulated by TNF‐α, IL‐10 and IFN‐γ. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.     

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.     

Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

Rapid release of cytoplasmic IL‐15 from tumor‐associated macrophages is an initial and critical event in IL‐12‐initiated tumor regression

This study reveals that the IL‐15 rapidly released into serum upon IL‐12 injection into tumor‐bearing mice is critical for the subsequent leukocytic infiltration of the tumor and tumor‐bearing tissue. The increase in serum IL‐15 occurs within 2 h after IL‐12 injection concomitantly with a decrease in cytoplasmic IL‐15 in tumor‐associated M? (TAM). Injection of anti‐IL‐15 one hour prior to IL‐12 abrogates subsequent leukocytic infiltration into the tumor and prevents the IL‐12‐induced reduction of primary tumor mass and the clearance of metastases. Administration of anti‐IL‐15 18 h after IL‐12 did not have a detectable impact on IL‐12‐induced leukocytic infiltration of the tumor. Deletion of NK cells had no impact on the IL‐12‐induced change in the functional phenotype of TAM or on the subsequent initiation of leukocytic infiltration of the tumor. In concert with our previous studies demonstrating that IL‐12 reduces tumor‐supportive activities of TAM, the current study supports the hypothesis that functional re‐programming of TAM not only undermines M? support for tumor growth but also contributes to a critical step in the initiation of anti‐tumor immune responses. In this context, the functional plasticity and pro‐immunogenic potential of TAM may constitute a significant and unappreciated target in existing cytokine therapies.     

Influence of the IL‐1Ra gene polymorphism on in vivo synthesis of IL‐1Ra and IL‐1β after live yellow fever vaccination

The presentation of hypogammaglobulinaemia in young males without a family history of immunodeficiency can pose a diagnostic problem. In the past, the presence of B-cells has suggested a diagnosis of common variable immunodeficiency (CVID), although genotypic analysis has now clarified that individuals with B cells may have mutations in their Btk gene. In order to address the issue of how many male individuals with a clinical diagnosis of CVID do in fact have mutations in the Btk gene, we analysed a group of 24 male patients. Single-strand conformation polymorphism (SSCP) analysis was used to screen the patient cohort for mutations in the Btk gene. Given the size of the Btk gene, the number of patients in the cohort and the amount of available DNA, multiplex PCR reactions were utilized to span the 19 exons and promoter region of the gene. Where abnormal migration patterns were observed with multiplex PCR reactions, in nine of the 24 patients, the individual Btk gene fragments were re-amplified and analysed again by SSCP. Following this analysis, four patients continued to demonstrate abnormal SSCP migration patterns. However, direct sequencing of the relevant Btk gene fragments for these four CVID patients revealed a mutation in only one patient. The mutation was the previously described polymorphism at position 2031 of Btk gene within exon 18. These results indicate that caution should be taken with the application of SSCP analysis to mutation detection. While it has a role to play in screening large patient cohorts, direct sequencing is a necessary adjunct to such analysis. Finally, the clinical diagnosis of CVID in this cohort successfully excluded males with Btk mutations.     

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.     

Inhibition of 4‐1BBL‐regulated TLR response in macrophages ameliorates endotoxin‐induced sepsis in mice

Activation of Toll‐like receptor (TLR) signaling rapidly induces the expression of inflammatory genes, which is persistent for a defined period of time. However, uncontrolled and excessive inflammation may lead to the development of diseases. 4‐1BB ligand (4‐1BBL) plays an essential role in sustaining the expression of inflammatory cytokines by interacting with TLRs during macrophage activation. Here, we show that inhibition of 4‐1BBL signaling reduced the inflammatory responses in macrophages and ameliorated endotoxin‐induced sepsis in mice. A 4‐1BB‐Fc fusion protein significantly reduced TNF production in macrophages by blocking the oligomerization of TLR4 and 4‐1BBL. Administration of 4‐1BB‐Fc suppressed LPS‐induced sepsis by reducing TNF production, and the coadministration of anti‐TNF and 4‐1BB‐Fc provided better protection against LPS‐induced sepsis. Taken together, these observations suggest that inhibition of the TLR/4‐1BBL complex formation may be highly efficient in protecting against continued inflammation, and that 4‐1BB‐Fc could be a potential therapeutic target for the treatment of inflammatory diseases.