PD-1 regulation of pathogenic IL-17-secreting γδ T cells in experimental autoimmune encephalomyelitis.

The PD-1-PD-L1 immune checkpoint helps to maintain self-tolerance and prevent the development of autoimmune diseases. Immune checkpoint inhibitors are successful immunotherapeutics for several cancers, but responding patients can develop immune-mediated adverse events. It is well established that PD-1 regulates CD4 and CD8 T-cell responses, but its role in controlling the activation of pathogenic γδ T cells is less clear. Here we examined the role of PD-1 in regulating γδ T cells in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We found that PD-1 was highly expressed on CD27- Vγ4 γδ T cells in the lymph node (LN) and CNS of mice with EAE. Treatment of mice with anti-PD-1 significantly augmented IL-17A-producing CD27- Vγ4 γδ T cells in the LN and CNS and enhanced the severity of EAE. The exacerbating effect of anti-PD-1 on EAE was lost in Tcrd-/- mice. Conversely, ligation of PD-1 suppressed Il17a and Rorc gene expression and IL-17A production by purified Vγ4 γδ T cells stimulated via the TCR, but not with IL-1ß and IL-23. Our study demonstrates that PD-1 regulates TCR-activated CD27- Vγ4 γδ T cells, but that cytokine-activated IL-17A producing γδ T cells escape the regulatory effects of the PD-1-PD-L1 pathway.

PYHIN protein IFI207 regulates cytokine transcription and IRF7 and contributes to the establishment of K. pneumoniae infection.

PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.

A population of proinflammatory T cells coexpresses αß and γδ T cell receptors in mice and humans.

T cells are classically recognized as distinct subsets that express αß or γδ TCRs. We identify a novel population of T cells that coexpress αß and γδ TCRs in mice and humans. These hybrid αß-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αß TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1ß and IL-23. Hybrid αß-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.

Interleukin-17A Serves a Priming Role in Autoimmunity by Recruiting IL-1ß-Producing Myeloid Cells that Promote Pathogenic T Cells.

Interleukin-17A (IL-17A) is a major mediator of tissue inflammation in many autoimmune diseases. Anti-IL-17A is an effective treatment for psoriasis and is showing promise in clinical trials in multiple sclerosis. In this study, we find that IL-17A-defective mice or mice treated with anti-IL-17A at induction of experimental autoimmune encephalomyelitis (EAE) are resistant to disease and have defective priming of IL-17-secreting γδ T (γδT17) cells and Th17 cells. However, T cells from Il17a-/- mice induce EAE in wild-type mice following in vitro culture with autoantigen, IL-1ß, and IL-23. Furthermore, treatment with IL-1ß or IL-17A at induction of EAE restores disease in Il17a-/- mice. Importantly, mobilization of IL-1ß-producing neutrophils and inflammatory monocytes and activation of γδT17 cells is reduced in Il17a-/- mice. Our findings demonstrate that a key function of IL-17A in central nervous system (CNS) autoimmunity is to recruit IL-1ß-secreting myeloid cells that prime pathogenic γδT17 and Th17 cells.

Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity.

Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate during glycolysis. The PK isoform PKM2 has additional roles in regulation of gene transcription and protein phosphorylation. PKM2 has been shown to control macrophage metabolic remodeling in inflammation, but its role in T cell biology is poorly understood. Here, we report PKM2 upregulation, phosphorylation, and nuclear accumulation in murine and human CD4+ T cells following activation in vitro. Treatment of T cells with TEPP-46, an allosteric activator that induces PKM2 tetramerization and blocks its nuclear translocation, strongly reduces their activation, proliferation, and cytokine production by inhibiting essential signaling pathways and thus preventing the engagement of glycolysis. TEPP-46 limits the development of both T helper 17 (Th17) and Th1 cells in vitro and ameliorates experimental autoimmune encephalomyelitis (EAE) in vivo. Overall, our results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation.

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1ß and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1-/- mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.

Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease.

The transcription factor BMAL1 is a core component of the molecular clock, regulating biological pathways that drive 24 h (circadian) rhythms in behaviour and physiology. The molecular clock has a profound influence on innate immune function, and circadian disruption is linked with increased incidence of multiple sclerosis (MS). However, the mechanisms underlying this association are unknown. Here we show that BMAL1 and time-of-day regulate the accumulation and activation of various immune cells in a CNS autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). In myeloid cells, BMAL1 maintains anti-inflammatory responses and reduces T cell polarization. Loss of myeloid BMAL1 or midday immunizations to induce EAE create an inflammatory environment in the CNS through expansion and infiltration of IL-1ß-secreting CD11b+Ly6Chi monocytes, resulting in increased pathogenic IL-17+/IFN-γ+ T cells. These findings demonstrate the importance of the molecular clock in modulating innate and adaptive immune crosstalk under autoimmune conditions.

A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.

The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1ß (IL-1ß) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.

Retinoic acid expression associates with enhanced IL-22 production by γδ T cells and innate lymphoid cells and attenuation of intestinal inflammation.

Retinoic acid (RA), a vitamin A metabolite, modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1ß or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition, RA treatment enhanced production of the IL-22-responsive antimicrobial peptides Reg3ß and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti-IL-22 neutralizing antibody, demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved, we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells.

Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine.

Whooping cough caused by Bordetella pertussis is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with B. pertussis and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with B. pertussis or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1ß-induced IL-17A production, but also reveal that optimum protection against B. pertussis requires induction of Th1, but not Th2 cells.

Therapeutic potential of targeting IL-17.

A June 2012 meeting in Dublin, Ireland, showcased advances in the understanding of the role of IL-17 and related cytokines in host immunity and how these cytokines have been successfully targeted for the treatment of autoimmunity.

IL-17-producing γδ T cells and innate lymphoid cells.

The inflammatory cytokine IL-17 plays a critical role in immunity to infection and is involved in the inflammatory pathology associated with certain autoimmune diseases, such as psoriasis and rheumatoid arthritis. While CD4(+) and CD8(+) T cells are important sources of this cytokine, recent evidence has suggested that γδ T cells and a number of families of innate lymphoid cells (ILCs) can secrete IL-17 and related cytokines. The production of IL-17 by γδ T cells appears to be largely independent of T-cell receptor activation and is promoted through cytokine signalling, in particular by IL-23 in combination with IL-1ß or IL-18. Therefore IL-17-secreting γδ T cells can be categorised as a family of cells similar to innate-like lymphoid cells. IL-17-secreting γδ T cells function as a part of mucosal defence against infection, with most studies to date focusing on their response to bacterial pathogens. γδ T cells also play a pathological role in certain autoimmune diseases, where they provide an early source of IL-17 and IL-21, which initiate responses mediated by conventional IL-17-secreting CD4(+) T cells (Th17 cells). ILCs lack an antigen receptor or other lineage markers, and ILC subsets that express the transcriptional factor RORγt have been found to secrete IL-17. Evidence is emerging that these newly recognised sources of IL-17 play both pathological and protective roles in inflammatory diseases as discussed in this article.

Interleukin-1 accounts for intrarenal Th17 cell activation during ureteral obstruction.

Interleukin 17A-secreting T-helper 17 (Th17) cells are pathogenic in inflammatory kidney diseases, but their intrarenal regulation is poorly understood. In order to better define Th17 cell dynamics during interstitial inflammation, we utilized the mouse unilateral ureteral obstruction model to analyze inflammatory cell subtypes by multicolor flow cytometry and cell sorting and by effects on in vitro-generated Th17 cells. Interleukin 17A expression localized to CCR6(+)CCR4(+/-)CD4(+) T-cells and progressively increased in obstructed kidneys. The number of CCR6(+)CD4(+) T-cells increased over 10-fold by 72 h, were enriched for interleukin 17A production, and were highly proliferative based on in vivo bromodeoxyuridine incorporation. Secreted products of leukocytes isolated from obstructed kidneys enhanced the interleukin 17A production of in vitro-generated Th17 cells. This Th17-enhancing activity was identified as interleukin-1 produced by renal dendritic cells and monocytes. The in vivo validity of these findings was confirmed in mice lacking the interleulin-1 receptor and in mice treated with a recombinant interleukin-1 receptor antagonist, each of which exhibited reduced intrarenal Th17 activity compared with control mice. Thus, the inflamed kidney accumulates CCR6(+) Th17 cells that undergo activation and proliferation. Production of interleukin 1 family cytokines by resident dendritic cells and infiltrating monocytes enhances intrarenal Th17 activation in acute kidney injury.

Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production.

Escherichia coli heat-labile enterotoxin (LT) is a powerful mucosal adjuvant; however, it is associated with toxic effects when delivered intranasally, and its mechanism of action is poorly understood. In this article, we demonstrate that LT acts as a highly effective adjuvant when administered parenterally, promoting Ag-specific IL-17, as well as IFN-γ, IL-4, and IL-10 production in response to coadministered Ags. We found that the adjuvant activity of LT was mediated in part by inducing dendritic cell (DC) activation; LT promoted CD80 and CD86 expression by DCs and enhanced IL-1α, IL-1ß, and IL-23 production. An LT mutant, LTK63, that lacks enzyme activity was less effective than the wild-type toxin in promoting DC maturation and the development of Ag-specific Th17 cells. LT enhanced IL-23 and IL-1α production from DCs via activation of ERK MAPK and IL-1ß secretion through activation of caspase-1 and the NLRP3 inflammasome. These cytokines played a major role in promoting Th17 responses by LT and LTK63. The induction of Th17 cells in vivo in response to LT and LTK63 as adjuvants was significantly reduced in IL-1RI-deficient mice. Finally, using a murine respiratory infection model, we demonstrated that LT can act as a highly effective adjuvant for a pertussis vaccine, promoting Ag-specific Th17 cells and protection against Bordetella pertussis challenge, which was significantly reduced in IL-17-defective mice. Our findings provide clear evidence that LT can promote protective immune responses in part through induction of innate IL-1 and, consequently, Th17 cells.

Caspase-1-processed cytokines IL-1beta and IL-18 promote IL-17 production by gammadelta and CD4 T cells that mediate autoimmunity.

IL-1ß plays a critical role in promoting IL-17 production by γδ and CD4 T cells. However, IL-1-targeted drugs, although effective against autoinflammatory diseases, are less effective against autoimmune diseases. Conversely, gain-of-function mutations in the NLRP3 inflammasome complex are associated with enhanced IL-1ß and IL-18 production and Th17 responses. In this study, we examined the role of caspase-1-processed cytokines in IL-17 production and in induction of experimental autoimmune encephalomyelitis (EAE). Killed Mycobacterium tuberculosis, the immunostimulatory component in CFA used for inducing EAE, stimulated IL-1ß and IL-18 production by dendritic cells through activation of the inflammasome complex and caspase-1. Dendritic cells stimulated with M. tuberculosis and myelin oligodendrocyte glycoprotein promoted IL-17 production by T cells and induced EAE following transfer to naive mice, and this was suppressed by a caspase-1 inhibitor and reversed by administration of IL-1ß or IL-18. Direct injection of the caspase-1 inhibitor suppressed IL-17 production by CD4 T cells and γδ T cells in vivo and attenuated the clinical signs of EAE. γδ T cells expressed high levels of IL-18R and the combination of IL-18 and IL-23, as with IL-1ß and IL-23, stimulated IL-17 production by γδ T cells, but also from CD4 T cells, in the absence of TCR engagement. Our findings demonstrate that caspase-1-processed cytokines IL-1ß and IL-18 not only promote autoimmunity by stimulating innate IL-17 production by T cells but also reveal redundancy in the functions of IL-1ß and IL-18, suggesting that caspase-1 or the inflammasome may be an important drug target for autoimmune diseases.

Regulation of interleukin-1beta by interferon-gamma is species specific, limited by suppressor of cytokine signalling 1 and influences interleukin-17 production.

Reports describing the effect of interferon-gamma (IFNgamma) on interleukin-1beta (IL-1beta) production are conflicting. We resolve this controversy by showing that IFNgamma potentiates IL-1beta release from human cells, but transiently inhibits the production of IL-1beta from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL-1beta and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNgamma and IFNbeta are anti-inflammatory. We observed that these cytokines suppress IL-1beta production in response to MTB, resulting in a reduced number of IL-17-producing cells. In human cells, IFNgamma increased IL-1beta production, and this might explain why IFNgamma is detrimental for multiple sclerosis. In mice, IFNgamma decreased IL-1beta and subsequently IL-17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.

Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis.

Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity.

Th17 cells, CD4(+) T cells that secrete interleukin-17 (IL-17), are pathogenic in autoimmune diseases and their development and expansion is driven by the cytokines IL-6, TGF-beta, IL-21, IL-1, and IL-23. However, there are also innate sources of IL-17. Here, we show that gammadelta T cells express IL-23R and the transcription factor RORgammat and produce IL-17, IL-21, and IL-22 in response to IL-1beta and IL-23, without T cell receptor engagement. IL-17-producing gammadelta T cells were found at high frequency in the brain of mice with experimental autoimmune encephalomyelitis (EAE). gammadelta T cells activated by IL-1beta and IL-23 promoted IL-17 production by CD4(+) T cells and increased susceptibility to EAE, suggesting that gammadelta T cells act in an amplification loop for IL-17 production by Th17 cells. Our findings demonstrate that gammadelta T cells activated by IL-1beta and IL-23 are an important source of innate IL-17 and IL-21 and provide an alternative mechanism whereby IL-1 and IL-23 may mediate autoimmune inflammation.

Inhibition of ERK MAPK suppresses IL-23- and IL-1-driven IL-17 production and attenuates autoimmune disease.

IL-17-producing CD4(+) T (Th17) cells are pathogenic in many autoimmune diseases. The induction and expansion of Th17 cells is directed by cytokines, including IL-23 and IL-1beta, produced by innate immune cells through activation of pathogen recognition receptors. The NF-kappaB and IFN regulatory factor families of transcriptional factors mediate IL-12 production; however, distinct signaling pathways appear to be required for IL-23 production. In this study, we show that inhibition of ERK MAPK suppressed IL-23 and IL-1beta production by dendritic cells stimulated with TLR or dectin-1 agonists but did not affect IL-12p70 production. Furthermore, an ERK inhibitor suppressed the ability of Ag-pulsed TLR-activated dendritic cells to induce Ag-specific Th17 cells in vivo, but interestingly also inhibited the induction of Th1 cells. Treatment with an ERK inhibitor attenuated experimental autoimmune encephalomyelitis (EAE), when administered either at the induction phase of acute EAE or during remission in the relapsing-remitting EAE model. This was associated with significant suppression of autoantigen-specific Th17 and Th1 responses. The suppressive effect of the ERK inhibitor on attenuation of EAE was reversed by administration of IL-1beta and IL-23. Our findings suggest that ERK MAPK plays a critical and hitherto undescribed role in activating innate production of IL-23 and IL-1beta, which promote pathogenic T cell responses, and therefore represents an important target for therapeutic intervention against autoimmune diseases.