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.     

IL‐33 activates eosinophils of visceral adipose tissue both directly and via innate lymphoid cells

Eosinophils are multifunctional leukocytes involved in allergic reactions as well as adipose tissue regulation. IL‐5 is required for eosinophil survival; however, the in vivo mechanisms of eosinophil regulation are not fully understood. A tg mouse model with il5 promoter‐driven EGFP expression was established for detecting the IL‐5‐producing cells in vivo. Il5‐egfp tg mice expressed high levels of EGFP in gonadal adipose tissue (GAT) cells. EGFP⁺ cells in GAT were mainly group 2 innate lymphoid cells (ILCs). IL‐33 preferentially expanded EGFP⁺ cells and eosinophils in GAT in vivo. EGFP⁺ ILCs were found to upregulate prg2 mRNA expression in GAT eosinophils. These results demonstrate that ILCs activate eosinophils in GAT. The blockage of IL‐33Rα, on the other hand, did not impair EGFP⁺ ILC numbers but did impair eosinophil numbers in vivo. GAT eosinophils expressed IL‐33Rα and IL‐33 expanded eosinophil numbers in CD90⁺ cell‐depleted mice. IL‐33 was further observed to induce the expression of retnla and epx mRNA in eosinophils. These findings demonstrate that IL‐33 directly activates eosinophils in GAT, and together with our other findings described above, our findings show that IL‐33 has dual pathways via which it activates eosinophils in vivo: a direct activation pathway and a group 2 ILC‐mediated pathway.     

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

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

Viral 5′‐triphosphate RNA and non‐CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR‐independent immune responses

Certain viral nucleic acids aggravate autoimmunity through nucleic acid‐specific TLR. Viral 5′‐triphosphate RNA (3P‐RNA) and double‐stranded non‐CpG DNA induce antiviral immunity via TLR‐independent pathways but their role in autoimmunity is unknown. Transient exposure of 16‐wk‐old MRL^lpr/lpr mice to 3P‐RNA aggravated lupus nephritis by increasing IFN signaling and decreasing CD4⁺CD25⁺ T cells. By contrast, transient exposure to non‐CpG DNA exacerbate lupus nephritis in association with splenomegaly, lymphoproliferation, hypergammaglobulinaemia and increased B220⁺CD138⁺ plasma cells. Both, 3P‐RNA and non‐CpG DNA increased glomerular complement factor C3c deposits but both nucleic acid formats were less potent in aggravating renal pathology as compared with CpG DNA. 3P‐RNA and non‐CpG DNA also localized to the glomerular mesangial cells and activated cultured mesangial cells to produce IL‐6. We conclude, 3P‐RNA or non‐CpG DNA both trigger autoimmune disease in MRL^lpr/lpr mice by specifically activating adaptive immunity but similarly enhance inflammation on the tissue level.     

Composition and functionality of the intrahepatic innate lymphoid cell‐compartment in human nonfibrotic and fibrotic livers

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue‐residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue‐resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44^? ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL‐13 when exposed to IL‐33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR‐3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.     

Parasites alter the pathological phenotype of lupus nephritis

Abstract

Lupus nephritis is one of the most serious complications of systemic lupus erythematosus and manifests with considerable phenotypic and histological heterogeneity. In particular, diffuse proliferative lupus nephritis (DPLN) and membranous lupus nephritis (MLN) represent morphologic forms that are polar opposites. DPLN is associated with autoimmune responses dominated by Th1 immune response associated with high levels of interferon (IFN)-γ. In contrast, a Th2 cytokine response is associated with the pathogenesis of MLN. MRL/lpr mice develop human LN-like immune complex-associated nephritis and provide a suitable histological model for human DPLN. Infection with Schistosoma mansoni skewed a Th2-type immune response induction and IL-10 in MRL/lpr mice, drastically changing the pathophysiology of glomerulonephritis from DPLN to MLN accompanied by increased IgG1 and IgE in the sera. T cells in 32-week-old MRL/lpr mice infected with S. mansoni expressed significantly more IL-4 and IL-10 than T cells of uninfected mice; T cells with IFN-γ were comparable between infected and uninfected MR/lpr mice. Thus, the helminthic infection modified the cytokine microenvironment and altered the pathological phenotype of autoimmune nephritis.     

ILC‐poiesis: Ensuring tissue ILC differentiation at the right place and time

Innate lymphoid cells (ILCs) represent a family of innate effector cells including NK cells, lymphoid tissue inducer (LTi) cells, and distinct ILC1, ILC2, and ILC3 subsets that produce IFN‐γ, IL‐5/IL‐13, and IL‐17A/IL‐22, respectively. ILCs accumulate at mucosal sites and can promote the first‐line defense against infection. ILCs are also implicated in tissue repair and can either pre‐empt, or alternatively, exacerbate inflammation. Studies in mice have identified ILC precursors in fetal liver and adult BM that have diverse lineage potential. As such, these sites have been considered as the ‘factories’ to generate mature ILC. Here, we summarize knowledge concerning murine and human ILC development and discuss the recent identification of circulating multipotent and unipotent ILC precursors. We propose an alternative model of “ILC‐poiesis”, whereby blood ILC precursors migrate into tissues to complete their differentiation into mature ILC subsets under the influence of local environmental factors. Within this framework, ILC‐poiesis guarantees appropriate ILC generation at the right place and the right time. We further discusss the potential applications of circulating ILC precursors for cell therapy of human disease.     

Reduced numbers of circulating group 2 innate lymphoid cells in patients with common variable immunodeficiency

Recent studies identified an emerging role of group 2 and 3 innate lymphoid cells (ILCs) as key players in the generation of T‐dependent and T‐independent antibody production. In this retrospective case‐control study, CD117⁺ ILCs (including the majority of ILC2 and ILC3) were reduced in patients with common variable immunodeficiency (CVID). The reduction in CD117⁺ ILCs was distinctive to CVID and could not be observed in patients with X‐linked agammaglobulinemia. Patients with a more pronounced reduction in CD117⁺ ILC numbers showed significantly lower numbers of peripheral MZ‐like B cells and an increased prevalence of chronic, non‐infectious enteropathy. Subsequent phenotyping of ILC subsets in CVID revealed that the reduction in CD117⁺ ILC numbers is due to a reduction in ILC2 numbers. In vitro expansion of CVID ILC2 in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired. Furthermore, upregulation of MHCII and IL‐2RA in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired in CVID ILC2. Thus, our results indicate a dysregulation of ILC subsets with a reduction in ILC2 numbers in CVID, however, further studies are needed to explore whether ILC abnormalities are a primary finding or secondary to disease complications encountered in CVID.     

CD47 deficiency ameliorates autoimmune nephritis in Faslpr mice by suppressing IgG autoantibody production

CD47, a self‐recognition marker, plays an important role in both innate and adaptive immune responses. To explore the potential role of CD47 in activation of autoreactive T and B cells and the production of autoantibodies in autoimmune disease, especially systemic lupus erythematosus (SLE), we have generated CD47 knockout Fas^lpr (CD47^?/??Fas^lpr) mice and examined histopathological changes in the kidneys, cumulative survival rates, proteinuria, extent of splenomegaly and autoantibodies, serum chemistry and immunological parameters. In comparison with Fas^lpr mice, CD47^?/??Fas^lpr mice exhibit a prolonged lifespan and delayed autoimmune nephritis, including glomerular cell proliferation, basement membrane thickening, acute tubular atrophy and vacuolization. CD47^?/??Fas^lpr mice have lower levels of proteinuria, associated with reduced deposition of complement C3 and C1q, and IgG but not IgM in the glomeruli, compared to age‐matched Fas^lpr mice. Serum levels of antinuclear antibodies and anti‐double‐stranded DNA antibodies are significantly lower in CD47^?/??Fas^lpr than in Fas^lpr mice. CD47^?/?–Fas^lpr mice also display less pronounced splenomegaly than Fas^lpr mice. The mechanistic studies further suggest that CD47 deficiency impairs the antigenic challenge‐induced production of IgG but not IgM, and that this effect is associated with reduction of T follicular cells and impairment of germinal centre development in lymphoid tissues. In conclusion, our results demonstrate that CD47 deficiency ameliorates lupus nephritis in Fas^lpr mice via suppression of IgG autoantibody production. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interleukin‐2 treatment reverses effects of cAMP‐responsive element modulator α‐over‐expressing T cells in autoimmune‐prone mice

Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self‐tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)‐2 critically maintains homeostasis of regulatory T cells (T_reg) and effector T cells in the periphery. Previously, we identified the cAMP‐responsive element modulator α (CREMα) as a major factor responsible for decreased IL‐2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over‐expresses CREMα in T cells (CD2CREMαtg), we provided in‐vivo evidence that CREMα indeed suppresses IL‐2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas^–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas^–/– mice. This was accompanied by a massive expansion of double‐negative (DN) T cells, enhanced numbers of interferon (IFN)‐γ‐producing T cells and reduced percentages of T_regs. Treatment of FVB/Fas^–/–CD2CREMαtg mice with IL‐2 restored the percentage of T_regs and reversed increased IFN‐γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL‐2 in vivo .     

Urtica dioica agglutinin,a Vβ8.3-specific superantigen,prevents the development of the systemic lupus erythematosus-like pathology of MRL lpr/lpr mice

The Vβ8.3-specific superantigenic lectin Urtica dioica agglutinin (UDA) was used to delete the Vβ8.3⁺ T cells in MRL lpr/lpr mice. In contrast to the systemic lupus erythematosus-like pathology which progresses with age in the phosphate-buffered saline-injected MRL lpr/lpr controls, UDA-treated animals did not develop overt clinical signs of lupus and nephritis. The pathogenic T cell clones thus reside within the Vβ8.3⁺ T cell population, which includes an expanded T cell clone described previously. Finally, UDA alters the production of autoantibodies in a sex-dependent manner.     

STS‐1 promotes IFN‐α induced autophagy by activating the JAK1‐STAT1 signaling pathway in B cells

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN‐α. IFN‐α induces autophagy via the JAK1‐STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS‐1 (suppressor of T‐cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS‐1 promoted IFN‐α‐induced autophagy in B cells by enhancing the JAK1‐STAT1 signaling activation. STS‐1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c‐cbl, and subsequently promoted IFN‐α‐induced phosphorylation of tyrosine kinase 2, leading to JAK1‐STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN‐α‐induced autophagy promoted by STS‐1, indicating that STS‐1 promotes IFN‐α‐induced autophagy via the JAK1‐STAT1 signaling. Our results demonstrate the importance of STS‐1 in regulating IFN‐α‐induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.     

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G‐CSF

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high‐risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft‐versus‐host disease and could also contribute to anti‐microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34⁺ cells are enriched in lymphoid‐committed precursors, while PB CD34⁺ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56⁺CD161⁺ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56⁺CD161⁺ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34⁺ cultures was lower for G‐CSF‐mobilized HSCs (good mobilizers) than for G‐CSF+plerixafor‐mobilized HSC (poor mobilizers). Moreover, G‐CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G‐CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.     

Lupus-prone MRL/faslpr/lpr mice display increased AID expression and extensive DNA lesions,comprising deletions and insertions,in the immunoglobulin locus: Concurrent upregulation of somatic hypermutation and class switch DNA recombination

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas^lpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas^lpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas^lpr/lpr B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas^lpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas^lpr/lpr mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas^lpr/lpr mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.