EGF-Receptor-Dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis.

Glomerulonephritis (GN) is a group of inflammatory diseases and an important cause of morbidity and mortality worldwide. The initiation of the inflammatory process is quite different for each type of GN; however, each GN is characterized commonly and variably by acute inflammation with neutrophils and macrophages and crescent formation, leading to glomerular death. Toll-like receptor (TLR) 7 is a sensor for self-RNA and implicated in the pathogenesis of human and murine GN. Here, we show that TLR7 exacerbates glomerular injury in nephrotoxic serum nephritis (NTN), a murine model of severe crescentic GN. TLR7-/- mice were resistant to NTN, although TLR7-/- mice manifested comparable immune-complex deposition to wild-type mice without significant defects in humoral immunity, suggesting that endogenous TLR7 ligands accelerate glomerular injury. TLR7 was expressed exclusively in macrophages in glomeruli in GN but not in glomerular resident cells or neutrophils. Furthermore, we discovered that epidermal growth factor receptor (EGFR), a receptor-type tyrosine kinase, is essential for TLR7 signaling in macrophages. Mechanistically, EGFR physically interacted with TLR7 upon TLR7 stimulation, and EGFR inhibitor completely blocked the phosphorylation of TLR7 tyrosine residue(s). EGFR inhibitor attenuated glomerular damage in wild-type mice, and no additional glomerular protective effects by EGFR inhibitor were observed in TLR7-/- mice. Finally, mice lacking EGFR in macrophages were resistant to NTN. This study clearly demonstrated that EGFR-dependent TLR7 signaling in macrophages is essential for glomerular injury in crescentic GN.

Monoallelic IRF5 deficiency in B cells prevents murine lupus.

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduced disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils did not reduce disease severity. B cell receptor and TLR7 signaling synergized to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increased B cell-intrinsic IL-6 and TNF-α production, both key requirements for germinal center (GC) responses, with IL-6 and TNF-α production in vitro and in vivo being substantially lower with loss of 1 allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation was reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression was dynamically regulated in vivo with increased expression in GC B cells compared with non-GC B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells was required to promote disease in murine lupus.

Inhibition of IRF4 in dendritic cells by PRR-independent and -dependent signals inhibit Th2 and promote Th17 responses.

Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.

c-Cbl targets PD-1 in immune cells for proteasomal degradation and modulates colorectal tumor growth.

Casitas B lymphoma (c-Cbl) is an E3 ubiquitin ligase and a negative regulator of colorectal cancer (CRC). Despite its high expression in immune cells, the effect of c-Cbl on the tumor microenvironment remains poorly understood. Here we demonstrate that c-Cbl alters the tumor microenvironment and suppresses Programmed cell death-1 (PD-1) protein, an immune checkpoint receptor. Using syngeneic CRC xenografts, we observed significantly higher growth of xenografts and infiltrating immune cells in c-Cbl+/- compared to c-Cbl+/+ mice. Tumor-associated CD8+ T-lymphocytes and macrophages of c-Cbl+/- mice showed 2-3-fold higher levels of PD-1. Functionally, macrophages from c-Cbl+/- mice showed a 4-5-fold reduction in tumor phagocytosis, which was restored with an anti-PD-1 neutralizing antibody suggesting regulation of PD-1 by c-Cbl. Further mechanistic probing revealed that C-terminus of c-Cbl interacted with the cytoplasmic tail of PD-1. c-Cbl destabilized PD-1 through ubiquitination- proteasomal degradation depending on c-Cbl's RING finger function. This data demonstrates c-Cbl as an E3 ligase of PD-1 and a regulator of tumor microenvironment, both of which were unrecognized components of its tumor suppressive activity. Advancing immune checkpoint and c-Cbl biology, our study prompts for probing of PD-1 regulation by c-Cbl in conditions driven by immune checkpoint abnormalities such as cancers and autoimmune diseases.

The immunomodulatory parasitic worm product ES-62 reduces lupus-associated accelerated atherosclerosis in a mouse model.

ES-62 is an anti-inflammatory phosphorylcholine-containing glycoprotein secreted by the filarial nematode Acanthocheilonema viteae. Accelerated atherosclerosis frequently occurs in systemic lupus erythematosus, resulting in substantial cardiovascular morbidity and mortality. We examined the effects of ES-62 in the gld.apoE(-/-) mouse model of this condition. Treatment with ES-62 did not substantially modulate renal pathology but caused decreased anti-nuclear autoantibody levels. Moreover, a striking 60% reduction in aortic atherosclerotic lesions was observed, with an associated decrease in macrophages and fibrosis. We believe that these latter findings constitute the first example of a defined parasitic worm product with therapeutic potential in atherosclerosis: ES-62-based drugs may represent a novel approach to control accelerated atherosclerosis in systemic lupus erythematosus.

CpGB DNA activates dermal macrophages and specifically recruits inflammatory monocytes into the skin.

Toll-like receptor 9 (TLR9) drives innate immune responses after recognition of foreign or endogenous DNA containing unmethylated CpG motifs. DNA-mediated TLR9 activation is highly implicated in the pathogenesis of several autoimmune skin diseases, yet its contribution to the inflammation seen in these diseases remains unclear. In this study, TLR9 ligand, CpGB DNA, was administered to mice via a subcutaneous osmotic pump with treatment lasting 1 or 4 weeks. Gene expression and immunofluorescence analyses were used to determine chemokine expression and cell recruitment in the skin surrounding the pump outlet. CpGB DNA skin treatment dramatically induced a marked influx of CD11b+ F4/80+ macrophages, increasing over 4 weeks of treatment, and induction of IFNγ and TNFα expression. Chemokines, CCL2, CCL4, CCL5, CXCL9 and CXCL10, were highly induced in CpGB DNA-treated skin, although abrogation of these signalling pathways individually did not alter macrophage accumulation. Flow cytometry analysis showed that TLR9 activation in the skin increased circulating CD11b+ CD115+ Ly6C(hi) inflammatory monocytes following 1 week of CpGB DNA treatment. Additionally, skin-resident CD11b+ cells were found to initially take up subcutaneous CpGB DNA and propagate the subsequent immune response. Using diphtheria toxin-induced monocyte depletion mouse model, gene expression analysis demonstrated that CD11b+ cells are responsible for the CpGB DNA-induced cytokine and chemokine response. Overall, these data demonstrate that chronic TLR9 activation induces a specific inflammatory response, ultimately leading to a striking and selective accumulation of macrophages in the skin.

Inhibition of type 4 cyclic nucleotide phosphodiesterase blocks intracellular TLR signaling in chronic lymphocytic leukemia and normal hematopoietic cells.

A subset of chronic lymphocytic leukemia (CLL) BCRs interacts with Ags expressed on apoptotic cells, suggesting that CLL BCRs have the potential to internalize apoptotic cell RNA- or DNA-containing fragments with resultant activation of TLR7 or TLR9, respectively. By blocking cAMP degradation, type 4 cAMP phosphodiesterase (PDE4) inhibitors activate cAMP-mediated signaling and induce apoptosis in CLL cells. In this study, we show that autologous irradiated leukemic cells induce proliferation in CLL cells and that such proliferation is blocked by a TLR7/8/9 inhibitor, by DNase, and by the PDE4 inhibitor rolipram. Rolipram also inhibited CLL cell proliferation induced by synthetic TLR7 and TLR9 agonists, as well as TLR agonist-induced costimulatory molecule expression and TNF-a (but not IL-6 or IL-10) production. Whereas treatment with a TLR9 agonist protected IgH V region unmutated, but not mutated, CLL cells from apoptosis, PDE4 inhibitors augmented apoptosis in both subtypes, suggesting that cAMP-mediated signaling may abrogate a TLR9-mediated survival signal in prognostically unfavorable IGHV unmutated CLL cells. Rolipram inhibited both TLR7/8- and TLR9-induced IFN regulatory factor 5 and NF-kB p65 nuclear translocation. PDE4 inhibitors also blocked TLR signaling in normal human immune cells. In PBMC and CD14-positive monocytes, PDE4 inhibitors blocked IFN-a or TNF-a (but not IL-6) production, respectively, following stimulation with synthetic TLR agonists or RNA-containing immune complexes. These results suggest that PDE4 inhibitors may be of clinical utility in CLL or autoimmune diseases that are driven by TLR-mediated signaling.

Interferon regulatory factor-5 deficiency ameliorates disease severity in the MRL/lpr mouse model of lupus in the absence of a mutation in DOCK2.

Interferon regulatory factor 5 (IRF5) polymorphisms are strongly associated with an increased risk of developing the autoimmune disease systemic lupus erythematosus. In mouse lupus models, IRF5-deficiency was shown to reduce disease severity consistent with an important role for IRF5 in disease pathogenesis. However these mouse studies were confounded by the recent demonstration that the IRF5 knockout mouse line contained a loss-of-function mutation in the dedicator of cytokinesis 2 (DOCK2) gene. As DOCK2 regulates lymphocyte trafficking and Toll-like receptor signaling, this raised the possibility that some of the protective effects attributed to IRF5 deficiency in the mouse lupus models may instead have been due to DOCK2 deficiency. We have therefore here evaluated the effect of IRF5-deficiency in the MRL/lpr mouse lupus model in the absence of the DOCK2 mutation. We find that IRF5-deficient (IRF5-/-) MRL/lpr mice develop much less severe disease than their IRF5-sufficient (IRF5+/+) littermates. Despite markedly lower serum levels of anti-nuclear autoantibodies and reduced total splenocyte and CD4+ T cell numbers, IRF5-/- MRL/lpr mice have similar numbers of all splenic B cell subsets compared to IRF5+/+ MRL/lpr mice, suggesting that IRF5 is not involved in B cell development up to the mature B cell stage. However, IRF5-/- MRL/lpr mice have greatly reduced numbers of spleen plasmablasts and bone marrow plasma cells. Serum levels of B lymphocyte stimulator (BLyS) were markedly elevated in the MRL/lpr mice but no effect of IRF5 on serum BLyS levels was seen. Overall our data demonstrate that IRF5 contributes to disease pathogenesis in the MRL/lpr lupus model and that this is due, at least in part, to the role of IRF5 in plasma cell formation. Our data also suggest that combined therapy targeting both IRF5 and BLyS might be a particularly effective therapeutic approach in lupus.

The effect of mycophenolate mofetil on disease development in the gld.apoE (-/-) mouse model of accelerated atherosclerosis and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by autoantibody production and inflammatory disease involving multiple organs. Premature atherosclerosis is a common complication of SLE and results in substantial morbidity and mortality from cardiovascular disease (CVD). The reasons for the premature atherosclerosis in SLE are incompletely understood, although chronic inflammation is thought to play an important role. There is currently no known preventative treatment of premature atherosclerosis in SLE. Mycophenolate mofetil (MMF) is an immunosuppressive agent that is commonly used for treatment of patients with SLE. In order to study the impact of this drug on murine lupus disease including premature atherosclerosis development, we treated gld.apoE(-/-) mice, a model of SLE and accelerated atherosclerosis, with MMF. We maintained seven-week old gld.apoE(-/-) mice on a high cholesterol Western diet with or without MMF. After 12 weeks on diet, mice receiving MMF showed decreased atherosclerotic lesion area compared to the control group. MMF treatment also improved the lupus phenotype, indicated by a significant decrease circulating autoantibody levels and ameliorating lupus nephritis associated with this model. This data suggests that the effects of MMF on the immune system may not only be beneficial for lupus, but also for inflammation driving lupus-associated atherosclerosis.

Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production.

Dendritic cell (DC) activation by nucleic acid-containing IgG complexes is implicated in systemic lupus erythematosus (SLE) pathogenesis. However, it has been difficult to definitively examine the receptors and signaling pathways by which this activation is mediated. Because mouse FcgammaRs recognize human IgG, we hypothesized that IgG from lupus patients might stimulate mouse DCs, thereby facilitating this analysis. In this study, we show that sera and purified IgG from lupus patients activate mouse DCs to produce IFN-alpha, IFN-beta, and IL-6 and up-regulate costimulatory molecules in a FcgammaR-dependent manner. This activation is only seen in sera with reactivity against ribonucleoproteins and is completely dependent on TLR7 and the presence of RNA. As anticipated, IFN regulatory factor (IRF)7 is required for IFN-alpha and IFN-beta production. Unexpectedly, however, IRF5 plays a critical role in IFN-alpha and IFN-beta production induced not only by RNA-containing immune complexes but also by conventional TLR7 and TLR9 ligands. Moreover, DC production of IL-6 induced by these stimuli is dependent on a functional type I IFNR, indicating the need for a type I IFN-dependent feedback loop in the production of inflammatory cytokines. This system may also prove useful for the study of receptors and signaling pathways used by immune complexes in other human diseases.

B cells and dendritic cells from V kappa 8 light chain transgenic mice activate MRL-lpr/gld CD4+ T cells.

Autoreactive CD4+ T cells are required for full expression of disease in human systemic lupus erythematosus and in spontaneous murine lupus. However, the Ag specificity of these CD4+ T cells remains largely unknown. Rheumatoid factor (RF) B cells function as highly efficient APCs by taking up immune complexes (IC) and presenting IC constituents to T cells. We hypothesized that Ag-specific CD4+ T cells in lupus-prone mice could be identified by stimulating the CD4+ T cells with RF B cells from AM14 RF BCR transgenic mice pulsed with IC containing lupus-associated autoantibodies and autoantigens. This approach identified several independent T cell lines that proliferated robustly in response to IC-pulsed spleen cells from the AM14 RF BCR transgenic mice. However, these T cells did not recognize an IC constituent. Instead, these T cells recognized a determinant dependent on the inheritance of the transgene-encoded Vkappa8 L chain, most likely a neoantigen created by the insertion of the transgene into the genome. Additionally, although the precise nature of the neoantigen is not known, the T cells described in this report may provide a useful tool for examining the role of T cells in the RF autoantibody response.

DNA and RNA autoantigens as autoadjuvants.

AM14 B cells are a prototype for those low affinity autoreactive B cells that routinely mature as naïve cells in peripheral lymphoid tissues. These cells express a transgene-encoded receptor specific for IgG2a and can be effectively activated by immune complexes that incorporate either mammalian DNA or mammalian RNA that has been released from dead or dying cells. Activation depends on the ability of the B-cell receptor to deliver antigen to an internal vesicular compartment containing either Toll-like receptor-9 (TLR9) or TLR7. Since TLR9 and TLR7 are thought to recognize microbial DNA and RNA preferentially, it is important to determine under what conditions mammalian DNA and RNA become effective TLR ligands, and whether the determining factor is delivery or structure. This issue has been addressed by using IgG2a mAbs to deliver immune complexes preloaded with defined fragments of DNA or RNA, or by using modified ODNs/ORNs. The data demonstrate that only certain nucleic acid sequences or structures can induce autoreactive B-cell proliferation, even when delivery to the appropriate TLR compartment is facilitated by uptake through the B-cell receptor (BCR).

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement.

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603-607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837-847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-alpha, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatin-immunoglobulin G complexes.

Dendritic cell (DC) activation by nucleic acid-containing immunoglobulin (Ig)G complexes has been implicated in systemic lupus erythematosus (SLE) pathogenesis. However, the mechanisms responsible for activation and subsequent disease induction are not completely understood. Here we show that murine DCs are much more effectively activated by immune complexes that contain IgG bound to chromatin than by immune complexes that contain foreign protein. Activation by these chromatin immune complexes occurs by two distinct pathways. One pathway involves dual engagement of the Fc receptor FcgammaRIII and Toll-like receptor (TLR)9, whereas the other is TLR9 independent. Furthermore, there is a characteristic cytokine profile elicited by the chromatin immune complexes that distinguishes this response from that of conventional TLR ligands, notably the induction of BAFF and the lack of induction of interleukin 12. The data establish a critical role for self-antigen in DC activation and explain how the innate immune system might drive the adaptive immune response in SLE.

Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors.

Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.