Increased ILC3s associated with higher levels of IL‐1β aggravates inflammatory arthritis in mice lacking phagocytic NADPH oxidase

The role of redox regulation in immune‐mediated arthritis has been previously described. However, the relationship between innate immune cells, including innate lymphoid cells (ILCs) and phagocyte‐derived ROS, in this process remains unclear. Here, we characterize ILCs and measure the IL‐1 family cytokines along with other cytokines relevant to ILC functions and development in serum‐induced arthritic joints in wild type and phagocytic NADPH oxidase (NOX2)‐deficient Ncf1^?/? mice. We found more severe serum‐induced joint inflammation and increased NCR⁺ ILC3s in inflamed joints of Ncf1^?/? mice. Furthermore, in vitro stimulation with IL‐1β on Tbet⁺ ILC1s from joints facilitated their differentiation into ROR‐γt⁺ ILC3s. Moreover, treatment with IL‐1 antagonists effectively lowered the proportions of NCR⁺ ILC3s and IL‐17A producing ILC3s in Ncf1^?/? arthritic mice and ameliorated the joint inflammation. These results suggest that NOX2 is an essential regulator of ILC transdifferentiation and may mediate this process in a redox‐dependent manner through IL‐1β production in the inflammatory joint. Our findings shed important light on the role of ILCs in the initiation and progression in tissue inflammation and delineate a novel innate immune cell‐mediated pathogenic mechanism through which redox regulation may determine the direction of immune responses in joints.     

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.     

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.     

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.     

Pathogenic IL‐23 signaling is required to initiate GM‐CSF‐driven autoimmune myocarditis in mice

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL‐23 stimulation of CD4⁺ T cells is required only briefly at the initiation of GM‐CFS‐dependent cardiac autoimmunity. IL‐23 signal, acting as a switch, turns on pathogenicity of CD4⁺ T cells, and becomes dispensable once autoreactivity is established. Il23a^?/? mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL‐23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4⁺ T cells raised from Il23a^?/? donors. Thus, IL‐23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL‐23‐induced GM‐CSF mediates the pathogenicity of CD4⁺ T cells in EAM. The neutralization of GM‐CSF abrogated cardiac inflammation. However, sustained IL‐23 signaling is required to maintain IL‐17A production in CD4⁺ T cells. Despite inducing inflammation in Il23a^?/? recipients comparable to wild‐type (WT), autoreactive CD4⁺ T cells downregulated IL‐17A production without persistent IL‐23 signaling. This divergence on the controls of GM‐CSF‐dependent pathogenicity on one side and IL‐17A production on the other side may contribute to the discrepant efficacies of anti‐IL‐23 therapy in different autoimmune diseases.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

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.     

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

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

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.     

Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent

Host protection to helminth infection requires IL‐4 receptor α chain (IL‐4Rα) signalling and the establishment of finely regulated Th2 responses. In the current study, the role of IL‐4Rα‐responsive T cells in Schistosoma mansoni egg‐induced inflammation was investigated. Egg‐induced inflammation in IL‐4Rα‐responsive BALB/c mice was accompanied with Th2‐biased responses, whereas T‐cell‐specific IL‐4Rα‐deficient BALB/c mice (iLck^creIl4ra^?/lox) developed Th1‐biased responses with heightened inflammation. The proportion of Foxp3⁺ Treg in the draining LN of control mice did not correlate with the control of inflammation and was reduced in comparison to T‐cell‐specific IL‐4Rα‐deficient mice. This was due to IL‐4‐mediated inhibition of CD4⁺Foxp3⁺ Treg conversion, demonstrated in adoptively transferred Rag2^?/? mice. Interestingly, reduced footpad swelling in Il4ra^?/lox mice was associated with the induction of IL‐4 and IL‐10‐secreting CD4⁺CD25^?CD103⁺Foxp3^? cells, confirmed in S. mansoni infection studies. Transfer of IL‐4Rα‐responsive CD4⁺CD25^?CD103⁺ cells, but not CD4⁺CD25^high or CD4⁺CD25^?CD103^? cells, controlled inflammation in iLck^creIl4ra^?/lox mice. The control of inflammation depended on IL‐10, as transferred CD4⁺CD25^?CD103⁺ cells from IL‐10‐deficient mice were not able to effectively downregulate inflammation. Together, these results demonstrate that IL‐4 signalling in T cells inhibits Foxp3⁺ Treg in vivo and promotes CD4⁺CD25^?CD103⁺Foxp3^? cells that control S. mansoni egg‐induced inflammation via IL‐10.     

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.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

Boswellic acids reduce Th17 differentiation via blockade of IL‐1β‐mediated IRAK1 signaling

Interferon‐gamma producing CD4⁺ T (Th1) cells and IL‐17‐producing CD4⁺ T (Th17) cells are involved in the pathogenesis of several autoimmune diseases including multiple sclerosis. Therefore, the development of treatment strategies controlling the generation and expansion of these effector cells is of high interest. Frankincense, the resin from trees of the genus Boswellia, and particularly its prominent bioactive compound acetyl‐11‐keto‐β‐boswellic acid (AKBA), have potent anti‐inflammatory properties. Here, we demonstrate that AKBA is able to reduce the differentiation of human CD4⁺ T cells to Th17 cells, while slightly increasing Th2‐ and Treg‐cell differentiation. Furthermore, AKBA reduces the IL‐1β‐triggered IL‐17A release of memory Th17 cells. AKBA may affect IL‐1β signaling by preventing IL‐1 receptor‐associated kinase 1 phosphorylation and subsequently decreasing STAT3 phosphorylation at Ser727, which is required for Th17‐cell differentiation. The effects of AKBA on Th17 differentiation and IL‐17A release make the compound a good candidate for potential treatment of Th17‐driven diseases.     

Hypoxia inducible factor‐1α‐induced interleukin‐33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor‐1α (HIF‐1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC‐derived HIF‐1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF‐1α and IL‐33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)?33 were correlated positively with that of HIF‐1α. A HIF‐1α‐interacting element was identified in the promoter region of IL‐33, indicating that HIF‐1α activity regulates IL‐33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF‐1α‐dependent IL‐33 expression in IEC. Our data thus demonstrate that HIF‐1α‐dependent IL‐33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis.     

Th2 cell‐derived IL‐4/IL‐13 promote ILC2 accumulation in the lung by ILC2‐intrinsic STAT6 signaling in mice

Infection of mice with the gastrointestinal helminth Nippostrongylus brasiliensis elicits profound local proliferation and accumulation of type 2 innate lymphoid cells (ILC2s) in the lung. The regulation of ILC2 proliferation and accumulation in the lung is poorly understood. Using T cell‐specific IL‐4/IL‐13‐deficient mice, we demonstrate that IL‐4/IL‐13 secretion from Th2 cells promotes proliferation and expansion of the ILC2 population in the lung of N. brasiliensis‐infected mice. Competitive mixed BM chimeras containing normal and STAT6‐deficient ILC2s further indicated that ILC2s have to respond directly to IL‐4/IL‐13 for this effect while STAT6 is not required for IL‐13 production in ILC2s. In addition, expression of a constitutively active form of STAT6 in ILC2s was sufficient to promote their proliferation in uninfected mice. The expression of MHC class II in ILC2s appeared to be enhanced by STAT6 signaling supporting the concept that Th2 cells and ILC2s can communicate in an antigen‐dependent manner resulting in a Th2‐regulated accumulation of ILC2s in the lung during an acute type 2 immune response. Based on our observations, targeting the STAT6 pathway in ILC2s could help to develop new treatments to dampen ILC2 proliferation in the lung and thereby ameliorate ILC2‐mediated allergic inflammation.     

PD‐1 modulates steady‐state and infection‐induced IL‐10 production in vivo

Programmed death‐1 (PD‐1) plays an important role in mediating immune tolerance through mechanisms that remain unclear. Herein, we investigated whether PD‐1 prevents excessive host tissue damage during infection with the protozoan parasite, Toxoplasma gondii. Surprisingly, our results demonstrate that PD‐1‐deficient mice have increased susceptibility to T. gondii, with increased parasite cyst counts along with reduced type‐1 cytokine responses (IL‐12 and IFN‐γ). PD‐1^?/? DCs showed no cell intrinsic defect in IL‐12 production in vitro. Instead, PD‐1 neutralization via genetic or pharmacological approaches resulted in a striking increase in IL‐10 release, which impaired type‐1‐inflammation during infection. Our results indicate that the absence of PD‐1 increases IL‐10 production even in the absence of infection. Although the possibility that such increased IL‐10 protects against autoimmune damage is speculative, our results show that IL‐10 suppresses the development of protective Th1 immune response after T. gondii infection.     

NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.