Heat shock protein 90 associates with Toll‐like receptors 7/9 and mediates self‐nucleic acid recognition in SLE

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease, and disease activity is associated with serum IFN‐α level. Plasmacytoid dendritic cells (pDCs) sense microbial as well as self‐nucleic acids by TLRs 7 and 9 and produce a large amount of IFN‐α. Here, we show that heat shock protein 90 (Hsp90) associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Inhibition of Hsp90 by various approaches including the use of Hsp90 inhibitor, a geldanamycin derivative, suppressed the Hsp90 association with TLR7/9, which resulted in inhibition of IFN‐α production, leading to improvement of SLE symptoms in mice. Notably, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Furthermore, we demonstrated that serum Hsp90 detected in SLE patients binds to self‐DNA and/or anti‐DNA Ab, thus leading to stimulation of pDCs to produce IFN‐α. Our data demonstrate that Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid‐related autoimmune diseases.     

Sequence independent interferon‐α induction by multimerized phosphodiester DNA depends on spatial regulation of Toll‐like receptor‐9 activation in plasmacytoid dendritic cells

Single‐stranded versus multimeric phosphorothioate‐modified CpG oligodeoxynucleotides (ODNs) undergo differential endosomal trafficking upon uptake into plasmacytoid dendritic cells (pDCs), correlating with Toll‐like receptor‐9‐dependent pDC maturation/activation (single‐stranded B‐type CpG ODN) or interferon‐α (IFN‐α) induction (multimeric A‐type CpG ODN), respectively. As was recently shown, IFN‐α production, other than by CpG ODNs, can also be induced in a sequence‐independent manner by phosphodiester (PD) ODNs multimerized by 3′ poly‐guanosine (poly‐G) tails. We investigate here the type of endosomal trafficking responsible for IFN‐α induction by natural PD ODN ligands. We show that 3′ extension with poly‐G tails leads to multimerization of single‐stranded PD ODNs and to enhanced cellular uptake into pDCs. While monomeric PD ODNs, which induce CpG‐dependent Toll‐like receptor‐9 stimulation and pDC maturation/activation, localized to late endosomal/lysosomal compartments, the poly‐G multimerized PD ODNs, which induce CpG‐independent IFN‐α production, located to vesicles with a distinct, ‘early’ endosomal phenotype. We conclude that poly‐G‐mediated multimerization of natural PD ODNs allows for sequence‐independent, Toll‐like receptor‐9‐dependent IFN‐α induction in pDCs by combining three distinct effects: relative protection of sensitive PD ODNs from extracellular and intracellular DNase degradation, enhanced cellular uptake and preferential early endosomal compartmentation.     

Sca‐1 expression defines developmental stages of mouse pDCs that show functional heterogeneity in the endosomal but not lysosomal TLR9 response

Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity and were shown to be identical to previously described natural interferon (IFN)‐α‐producing cells. Here, we describe two functionally distinct pDC subpopulations that are characterized by the differential expression of stem cell antigen‐1 (Sca‐1; Ly‐6A/E). Sca‐1^? pDCs are mainly found in the BM, appear first during development, show a higher proliferative activity, and represent the more precursor phenotype. Sca‐1⁺ pDCs are mostly located in secondary lymphoid organs and represent a later developmental stage. Sca‐1^? pDCs give rise to an Sca‐1⁺ subset upon activation or in response to endogenous type I IFN. Interestingly, in contrast to Sca‐1^? pDCs, Sca‐1⁺ pDCs are defective in IFN‐α production upon endosomal TLR9 stimulation, whereas lysosomal signaling via TLR9 is functional in both subsets. Gene expression analysis revealed that osteopontin is strongly upregulated in Sca‐1^? pDCs. These data provide evidence for the molecular basis of the observed functional heterogeneity, as the intracellular isoform of osteopontin couples TLR9 signaling to IFN‐α expression. Taken together, our results indicate that Sca‐1^? pDCs are an early developmental stage of pDCs with distinct innate functions representing the true murine natural IFN‐α‐producing cells.     

Elevated serum interleukin (IL)-12p40 levels in common variable immunodeficiency disease and decreased peripheral blood dendritic cells: analysis of IL-12p40 and interferon-gamma gene

Common variable immunodeficiency disease (CVID) is a heterogeneous syndrome characterized by low immunoglobulin serum levels and recurrent bacterial infections. Several studies suggest that CVID patients have a polarized immune response towards a T helper type 1 phenotype (TH1). However, the factors causing the TH1 polarization remain to be determined in this disease. In the present study, serum interleukin (IL)-12, interferon (IFN)-gamma levels and the IL-12p40 and IFN-gamma gene were studied in CVID patients. Furthermore, we evaluate dendritic cells (DCs) compartment, myeloid dendritic cells (mDCs) and plasmocytoid dendritic cells (pDCs), which help to differentiate naive T cells preferentially into TH1 and TH2, respectively. The serum IL-12p40 subunit levels were increased significantly in CVID patients compared to healthy controls. We examined whether these elevated serum IL-12p40 levels are associated with IFN-gamma or IL-12p40 gene polymorphisms, or with new mutations in the IL-12p40 promoter gene. In our hands, no new mutations were found and gene polymorphisms frequencies in CVID patients were similar to the control population. In conclusion, the elevated serum levels of IL-12p40 found in our CVID patients were not related to these genetic variations. The DC compartment analysis did not show an imbalance between pDCs and mDCs, but revealed the presence of low numbers and percentage of both DC populations in CVID.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

IFN‐β therapy modulates B‐cell and monocyte crosstalk via TLR7 in multiple sclerosis patients

The implication of B lymphocytes in the immunopathology of multiple sclerosis (MS) is increasingly recognized. Here we investigated the response of B cells to IFN‐β, a first‐line therapy for relapsing‐remitting MS patients, upon stimulation with TLR. IFN‐β restored the frequency of TLR7‐induced IgM and IgG‐secreting cells in MS patients to the levels found in healthy donors, showing a specific deficiency in the TLR7 pathway. However, no difference was observed in the TLR9 response. Furthermore, in MS‐derived PBMCs, TLR7‐mediated production of IL‐6 and the ex vivo expression of B‐cell‐activating factor of the TNF family, two crucial cytokines for B‐cell differentiation and survival, were induced by IFN‐β. Depletion of monocytes, which are key producers of both IL‐6 and B‐cell‐activating factor of the TNF family, showed that TLR7‐mediated B‐cell differentiation into Ig‐secreting cells is strongly dependent on the cross‐talk between B cells and monocytes. Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and monocytes isolated from MS‐affected individuals as compared with those from healthy donors, which was rescued by IFN‐β therapy. Collectively, our data unveil a novel TLR7‐regulated mechanism in in vivo IFN‐β‐stimulated whole leukocytes that could be exploited to define new TLR7‐based strategies for the treatment of MS.     

Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN‐β production capable of improving dendritic cell function

Viral double‐stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine–polycytidylic acid (poly I:C) and polyadenylic–polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN‐β autocrine loop, dsRNA‐elicited IFN‐β production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN‐β at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte‐derived DCs. IFN‐β produced by poly I:C‐activated human cancer cells increased the capacity of monocyte‐derived DCs to stimulate IFN‐γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3^?/? mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1^?/? mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN‐β production and contribute to the antitumoral response.     

Proinflammatory cytokine gene single nucleotide polymorphisms in common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous group of primary immunodeficiency diseases. Cytokine production could be affected in CVID patients, whereas its alteration could be due to genetic polymorphisms within coding and promoter regions of the cytokine genes. This study was performed to analyse the proinflammatory cytokine single nucleotide polymorphisms in CVID. The allele and genotype frequencies of a number polymorphic genes coding tumour necrosis factor (TNF)‐α, interleukin (IL)‐1α, IL‐1β, IL‐1R, IL‐1RA and IL‐6 were investigated and compared between two groups of CVID patients and controls. The IL‐6 GA genotype at position nt565 was significantly over‐represented in the patient group (P < 0·001), while the IL‐6 GG genotype at position ?174 (P = 0·006) and the GG genotype at position nt565 (P < 0·001) were significantly lower than controls. The TNF‐α AG genotype at position ?308 in the patient group was increased significantly in comparison with controls (P = 0·027), but the GG genotype at the same position was significantly decreased (P = 0·011). IL‐6 CA and GA haplotypes were the most frequent haplotypes in the patients (P < 0·005), whereas TNF‐α GA (P = 0·002) and IL‐6 GG (P < 0·001) haplotypes were decreased significantly in the patients in comparison with controls. Cytokine single nucleotide polymorphisms could have a role in pathophysiology of CVID. High production of TNF‐α is expected in some CVID patients based on the frequency of genotypes/haplotypes of these cytokine gene polymorphisms.     

Critical roles of conventional dendritic cells in promoting T cell‐dependent hepatitis through regulating natural killer T cells

Dendritic cells (DCs) play critical roles in initiating and regulating innate immunity as well as adaptive immune responses. However, the role of conventional dendritic cells (cDCs) in concanavalin A (ConA)‐induced fulminant hepatitis is unknown. In this study, we demonstrated that depletion of cDCs using either CD11c‐diphtheria toxin receptor transgenic mice (DTR Tg) mice or anti‐CD11c antibody reduced the severity of liver injury significantly, indicating a detrimental role of cDCs in ConA‐induced hepatitis. We elucidated further the pathological role of cDCs as being the critical source of interleukin (IL)‐12, which induced the secretion of interferon (IFN)‐γ by natural killer (NK) T cells. Reconstitution of cDCs‐depleted mice with IL‐12 restored ConA‐induced hepatitis significantly. Furthermore, we determined that NK T cells were the target of DC‐derived IL‐12, and NK T cells contributed to liver inflammation and injury through production of IFN‐γ. In summary, our study demonstrated a novel function of cDCs in mediating ConA‐induced hepatitis through regulating IFN‐γ secretion of NK T cells in an IL‐12‐dependent fashion. Targeting cDCs might provide potentially therapeutic applications in treating autoimmune related liver diseases.     

TLR9 and STING agonists synergistically induce innate and adaptive type‐II IFN

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type‐1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K‐type) induces IFNs only weakly and STING ligands rather induce type‐2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type‐1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING‐ligand cyclic GMP–AMP (cGAMP), culminating in NK cell IFN‐γ (type‐II IFN) production, is due to the concurrent effects of IL‐12 and type‐I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type‐1 adjuvant, capable of inducing strong T_h1‐type responses, as demonstrated by enhanced antigen‐specific IgG2c and IFN‐γ production, as well as cytotoxic CD8⁺ T‐cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen‐free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type‐II IFN inducer.     

Properdin and factor H production by human dendritic cells modulates their T‐cell stimulatory capacity and is regulated by IFN‐γ

Dendritic cells (DCs) and complement are both key members of the innate and adaptive immune response. Recent experimental mouse models have shown that production of alternative pathway (AP) components by DCs strongly affects their ability to activate and regulate T‐cell responses. In this study we investigated the production and regulation of properdin (fP) and factor H (fH) both integral regulators of the AP, by DCs and tolerogenic DCs (tolDCs). Both fP and fH were produced by DCs, with significantly higher levels of both AP components produced by tolDCs. Upon activation with IFN‐γ both cells increased fH production, while simultaneously decreasing production of fP. IL‐27, a member of the IL‐12 family, increased fH, but production of fP remained unaffected. The functional capacity of fP and fH produced by DCs and tolDCs was confirmed by their ability to bind C3b. Inhibition of fH production by DCs resulted in a greater ability to induce allogenic CD4⁺ T‐cell proliferation. In contrast, inhibition of fP production led to a significantly reduced allostimulatory capacity. In summary, this study shows that production of fP and fH by DCs, differentially regulates their immunogenicity, and that the local cytokine environment can profoundly affect the production of fP and fH.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

IFN‐γ‐producing NKT cells exacerbate sepsis by enhancing C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT‐cell‐deficient mice of C57BL/6 background during cecal ligation and puncture‐induced sepsis. The levels of C5a, IFN‐γ, and IL‐10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d^?/? mice, while the mortality rate was lower in CD1d^?/? mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d^?/? mice. As assessed by intracellular staining, NKT cells expressed IFN‐γ, while neutrophils expressed IL‐10. Upon coculture, IL‐10‐deficient NKT cells enhanced IL‐10 production by WT, but not IFN‐γR‐deficient, neutrophils. Meanwhile, CD1d^?/? mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL‐10 administration into CD1d^?/? mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN‐γ‐deficient, NKT cells into CD1d^?/? mice suppressed CD55 expression on neutrophils, but increased IL‐10 and C5a levels. Taken together, IFN‐γ‐producing NKT cells enhance C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.     

Synergistic antiviral activity of Sofosbuvir and type‐I interferons (α and β) against Zika virus

Zika virus (ZIKV) is transmitted by mosquitoes and causes Dengue‐like illness, neurological symptoms such as Guillain‐Barré Syndrome and microcephaly in children born to infected pregnant mothers. Recently, the World Health Organization (WHO) declared ZIKV infection as a Global Health Emergency. However, there are no known prophylactic or therapeutic measures against this virus. As a proof of concept toward combination therapeutic strategy against ZIKV, combinations of host‐targeted (Interferon‐α and Interferon‐β) and direct acting (Sofosbuvir) antivirals were evaluated in a hepatic cell line (Huh7) using a Cytoprotection (CP) assay. The combination of these antivirals resulted in synergistic inhibition of ZIKV infection in the in vitro CP assay. Additional testing in a ZIKV yield assay demonstrated that combination treatment of these antivirals conferred >2‐log reduction in the release of viral RNA. Measurement of ZIKV proteins in the cells infected with multiple ZIKV strains isolated from different geographical regions (Americas, Asia, and Africa) using an immunofluorescence assay confirmed the effective antiviral activity of this combination against ZIKV. These results demonstrate the in vitro proof of concept (POC) for using a combination approach utilizing the strengths of both virus and host‐targeted antivirals. These results suggest the effectiveness of the combination strategy in combating ZIKV, in the in vitro systems. Further evaluation of such combination therapies in vivo might provide an impetus for the development of effective ZIKV therapeutic strategies.     

Differential effects of stromal derived factor‐1α (SDF‐1α) on early and late stages of human megakaryocytic development

Stromal derived factor‐1α (SDF‐1α), the high‐affinity ligand of CXC‐chemokine receptor 4 (CXCR4), was added to human CD34⁺ hematopoietic progenitor cells that can be induced to differentiate along the monocytic or megakaryocytic lineages. In control liquid cell cultures supplemented with two different cytokine cocktails: stem cell factor (SCF), interleukin‐3 (IL‐3), macrophage‐colony stimulating factor (M‐CSF), and 10% fetal calf serum (FCS), or, SCF and thrombopoietin (TPO), the expression of surface CXCR4 progressively increased in both the CD14⁺ monocytic and CD41⁺ megakaryocytic lineages. While SDF‐1α caused only modest effects on cells of the monocytic lineage, it induced profound down‐regulation of CXCR4 in megakaryocytic cells at all stages of differentiation. Moreover, while SDF‐1α initially up‐regulated the early megakaryocytic antigen CD41, at later time points (days 12–16) it induced down‐regulation of the late megakaryocytic antigen CD42b. Consistently, at day 16, the number of mature megakaryocytes was significantly decreased in cultures supplemented with SDF‐1α. These findings indicate that, besides its primary role in regulating the retention of precursor cells in hematopoietic tissues, the SDF‐1α/CXCR4 system participates in the regulation of megakaryocytic development by stimulating the formation of immature megakaryoblasts and inhibiting the formation of mature megakaryocytes. Anat Rec 260:141–147, 2000. © 2000 Wiley‐Liss, Inc.