Kerstin Nundel

Suppression of systemic autoimmunity by the innate immune adaptor STING

Significance Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that presents with a diverse array of clinical symptoms and afflicts over 1.5 million Americans. Current treatments involve immunosuppressive regimens associated with debilitating and adverse effects. With the description of a role for innate signaling in SLE, safe and efficient therapies that block Toll-like receptors also have been stymied by the relative short in vivo half lives of known inhibitors and the dangerous outcome of complete MyD88 blockade. Key natural regulators of the disease process are not well described but are more likely to provide disease-specific therapeutics with fewer adverse effects. In this study, we have identified a novel function for Stimulator of interferon genes as a suppressor of disease and a target for future SLE therapeutics. Cytosolic DNA-sensing pathways that signal via Stimulator of interferon genes (STING) mediate immunity to pathogens and also promote autoimmune pathology in DNaseII- and DNaseIII-deficient mice. In contrast, we report here that STING potently suppresses inflammation in a model of systemic lupus erythematosus (SLE). Lymphoid hypertrophy, autoantibody production, serum cytokine levels, and other indicators of immune activation were markedly increased in STING-deficient autoimmune-prone mice compared with STING-sufficient littermates. As a result, STING-deficient autoimmune-prone mice had significantly shorter lifespans than controls. Importantly, Toll-like receptor (TLR)-dependent systemic inflammation during 2,6,10,14-tetramethylpentadecane (TMPD)-mediated peritonitis was similarly aggravated in STING-deficient mice. Mechanistically, STING-deficient macrophages failed to express negative regulators of immune activation and thus were hyperresponsive to TLR ligands, producing abnormally high levels of proinflammatory cytokines. This hyperreactivity corresponds to dramatically elevated numbers of inflammatory macrophages and granulocytes in vivo. Collectively these findings reveal an unexpected negative regulatory role for STING, having important implications for STING-directed therapies.

Phenotype and function of B cells and dendritic cells from interferon regulatory factor 5-deficient mice with and without a mutation in DOCK2

Interferon regulatory factor 5-deficient (IRF5 (-/-) ) mice have been used for many studies of IRF5 biology. A recent report identifies a mutation in dedicator of cytokinesis 2 (DOCK2) as being responsible for the abnormal B-cell development phenotype observed in the IRF5 (-/-) line. Both dedicator of cytokinesis 2 (DOCK2) and IRF5 play important roles in immune cell function, raising the issue of whether immune effects previously associated with IRF5 are due to IRF5 or DOCK2. Here, we defined the insertion end-point of the DOCK2 mutation and designed a novel PCR to detect the mutation in genomic DNA. We confirmed the association of the DOCK2 mutation and the abnormal B-cell phenotype in our IRF5 (-/-) line and also established another IRF5 (-/-) line without the DOCK2 mutation. These two lines were used to compare the role of IRF5 in dendritic cells (DCs) and B cells in the presence or absence of the DOCK2 mutation. IRF5 deficiency reduces IFN-α, IFN-β and IL-6 production by Toll-like receptor 9 (TLR9)- and TLR7-stimulated DCs and reduces TLR7- and TLR9-induced IL-6 production by B cells to a similar extent in the two lines. Importantly however, IRF5 (-/-) mice with the DOCK2 mutation have higher serum levels of IgG1 and lower levels of IgG2b, IgG2a/c and IgG3 than IRF5 (-/-) mice without the DOCK2 mutation, suggesting that the DOCK2 mutation confers additional Th2-type effects. Overall, these studies help clarify the function of IRF5 in B cells and DCs in the absence of the DOCK2 mutation. In addition, the PCR described will be useful for other investigators using the IRF5 (-/-) mouse line.

Cell-Intrinsic Expression of TLR9 in Autoreactive B Cells Constrains BCR/TLR7-Dependent Responses

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9–double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

Cutting Edge: DNase II Deficiency Prevents Activation of Autoreactive B Cells by Double-Stranded DNA Endogenous Ligands

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b13d/3d DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus–prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II–mediated degradation of self-DNA is required for TLR9 activation.

TLR9 Deficiency Leads to Accelerated Renal Disease and Myeloid Lineage Abnormalities in Pristane-Induced Murine Lupus

Systemic lupus erythematosus (SLE) is a chronic, life-threatening autoimmune disorder, leading to multiple organ pathologies and kidney destruction. Analyses of numerous murine models of spontaneous SLE have revealed a critical role for endosomal TLRs in the production of autoantibodies and development of other clinical disease manifestations. Nevertheless, the corresponding TLR9-deficient autoimmune-prone strains consistently develop more severe disease pathology. Injection of BALB/c mice with 2,6,10,14-tetramethylpentadecane (TMPD), commonly known as pristane, also results in the development of SLE-like disease. We now show that Tlr9−/− BALB/c mice injected i.p. with TMPD develop more severe autoimmunity than do their TLR-sufficient cohorts. Early indications include an increased accumulation of TLR7-expressing Ly6Chi inflammatory monocytes at the site of injection, upregulation of IFN-regulated gene expression in the peritoneal cavity, and an increased production of myeloid lineage precursors (common myeloid progenitors and granulocyte myeloid precursors) in the bone marrow. TMPD-injected Tlr9−/− BALB/c mice develop higher autoantibody titers against RNA, neutrophil cytoplasmic Ags, and myeloperoxidase than do TMPD-injected wild-type BALB/c mice. The TMP-injected Tlr9−/− mice, and not the wild-type mice, also develop a marked increase in glomerular IgG deposition and infiltrating granulocytes, much more severe glomerulonephritis, and a reduced lifespan. Collectively, the data point to a major role for TLR7 in the response to self-antigens in this model of experimental autoimmunity. Therefore, the BALB/c pristane model recapitulates other TLR7-driven spontaneous models of SLE and is negatively regulated by TLR9.

The role of Bruton's tyrosine kinase in the development and BCR/TLR-dependent activation of AM14 rheumatoid factor B cells

The protein kinase Btk has been implicated in the development, differentiation, and activation of B cells through its role in the BCR and TLR signaling cascades. These receptors and in particular, the BCR and either TLR7 or TLR9 also play a critical role in the activation of autoreactive B cells by RNA‐ or DNA‐associated autoantigens. To explore the role of Btk in the development of autoreactive B cells, as well as their responses to nucleic acid‐associated autoantigens, we have now compared Btk‐sufficient and Btk‐deficient mice that express a prototypic RF BCR encoded by H‐ and L‐chain sdTgs. These B cells bind autologous IgG2a with low affinity and only proliferate in response to IgG2a ICs that incorporate DNA or RNA. We found that Btk‐sufficient RF+ B cells mature into naïve FO B cells, all of which express the Tg BCR, despite circulating levels of IgG2a. By contrast, a significant proportion of Btk‐deficient RF+ B cells acquires a MZ or MZ precursor phenotype. Remarkably, despite the complete inability of RF+ Xid/y B cells to respond to F(ab′)2 anti‐IgM, RF+ Xid/y B cells could respond well to autoantigen‐associated ICs. These data reveal unique features of the signaling cascades responsible for the activation of autoreactive B cells.

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27– human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-&ggr; promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Taking the STING out of TLR-driven autoimmune diseases: good, bad, or indifferent?

Both endosomal and cytosolic‐nucleic acid–sensing receptors can detect endogenous ligands and promote autoimmunity and autoinflammation. These responses involve a complex interplay among and between the cytosolic and endosomal sensors involving both hematopoietic and radioresistant cells. Cytosolic sensors directly promote inflammatory responses through the production of type I IFNs and proinflammatory cytokines. Inflammation‐associated tissue damage can further promote autoimmune responses indirectly, as receptor‐mediated internalization of the resulting cell debris can activate endosomal Toll‐like receptors (TLR). Both endosomal and cytosolic receptors can also negatively regulate inflammatory responses. A better understanding of the factors and pathways that promote and constrain autoimmune diseases will have important implications for the development of agonists and antagonists that modulate these pathways.

Synergy between Hematopoietic and Radioresistant Stromal Cells Is Required for Autoimmune Manifestations of DNase II−/−IFNaR−/− Mice

Detection of endogenous nucleic acids by cytosolic receptors, dependent on STING, and endosomal sensors, dependent on Unc93b1, can provoke inflammatory responses that contribute to a variety of autoimmune and autoinflammatory diseases. In DNase II–deficient mice, the excessive accrual of undegraded DNA leads to both a STING-dependent inflammatory arthritis and additional Unc93b1-dependent autoimmune manifestations, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. In this study, we use bone marrow chimeras to show that clinical and histological inflammation in the joint depends upon DNase II deficiency in both donor hematopoietic cells and host radioresistant cells. Additional features of autoimmunity in these mice, known to depend on Unc93b1 and therefore endosomal TLRs, also require DNase II deficiency in both donor and host compartments, but only require functional TLRs in the hematopoietic cells. Collectively, our data demonstrate a major role of both stromal and hematopoietic cells in all aspects of DNA-driven autoimmunity. These findings further point to the importance of cytosolic nucleic acid sensors in creating an inflammatory environment that facilitates the development of Unc93b1-dependent autoimmunity.

II-14 disturbed clearance of apoptotic debris in pristane-treated tlr9ko mice leads to accumulation of a unique macrophage population

Background Nucleic acid binding TLRs have been found to play a critical role in the production of autoantibodies and disease development in animal models of SLE. Intriguingly, TLR9 appears to play both a protective and disease promoting role. While TLR9 is required for the production of anti-dsDNA autoantibodies, TLR9KO autoimmune-prone mice develop more severe disease than their TLR9-sufficient counterparts. Studies from our group and others have pointed to B cell expression of TLR9 as a key determining factor. However, our recent studies point to an additional role for TLR9in myeloid lineage cells. Materials and methods Pristane injected BALB/c wildtype (WT) and TLR9KO mice were analysed for disease severity at 5 months. Kidney sections were stained for IgG deposition and Ly6G positive cells. Single cell suspensions of different tissues were analysed using flow cytometry. Mixed BM chimaeras (50% WT: 50% TLR9KO) were injected with pristane and myeloid populations were analysed. For apoptotic cell clearance, WT and TLR9KO bone marrow derived macrophages (BMDM) were stimulated with CFSE labelled apoptotic cells and analysed 24 hours later by confocal microscopy. Results We have found that TLR9-deficiency dramatically exacerbates the onset of renal disease resulting in decreased survival. Increased levels of IgG accumulate in pristane treated TLR9KO glomeruli compared to WT glomeruli, and the increased IgG deposits are associated with an increased myeloid infiltrate. Moreover, this myeloid infiltrate contained an increased frequency of granulocytes a well as an unusual CD11b+ Ly6Cint Ly6Gint (Ly6CGint) subset. To better understand the origin of these populations, the myeloid subsets of pristane-treated mixed (TLR9WT + TLR9KO) BM chimaeras were analysed. Remarkably, the Ly6CGint population was entirely derived from the TLR9KO stem cells. Morphologic analysis revealed that the Ly6CGint population are macrophages containing large lipid droplets, suggesting a role for TLR9 in degradation of pristane. Further in vitro analysis of BMDMs stimulated with apoptotic cells showed that most WT BMDMs cleared apoptotic cells by 24 h. However, a large fraction of TLR9-deficient BMDMs still had un-degraded apoptotic cells in the lysosomal compartment, suggesting a role for TLR9 in clearance. Conclusions These data demonstrate a direct effect of TLR9-deficiency on the expansion of a unique CD11b+ population, and further suggest that these cells play a major and direct role in the accelerated disease characteristic in TLR9KO mice. Furthermore, a specific role for TLR9 in the clearance of apoptotic cells may be the underlying cause for the accumulation of this CD11b+ subset.