Targeting Multiple End Organs in Lupus and Other Systemic Rheumatic Diseases by Inhibiting Bruton's Tyrosine Kinase.

Bruton tyrosine kinase (Btk) plays a vital role in activating and differentiating B-cells and regulating signaling in myeloid cells. Indeed, the potential use of Btk inhibitors in preventing lupus has been reported. Here, we extend these observations to 4 additional models of end-organ inflammation: (a) BWF1 lupus nephritis mice, (b) anti-GBM nephritis, (c) bleomycin-induced systemic sclerosis like skin disease, and (d) bleomycin-induced lung disease. In agreement with the previous studies, BTK inhibitor (BTKB66) treatment was effective in treating lupus nephritis in terms of reducing renal damage both functionally and histologically, accompanied by significant decrease in proteinuria. Both low-dose and high-dose BTKB66 profoundly blocked renal disease in the anti-GBM nephritis model, with efficacy that was comparable to that seen with dexamethasone. This study provides the first evidence that BTK inhibition has both therapeutic and preventative effects in bleomycin-induced SSc-like disease, in terms of reducing skin thickness, fibrosis, collagen deposition, and inflammation. Likewise, significantly lower lung inflammatory cell infiltration was observed after treatment with BTKB66. Therapeutic benefit was associated with lower numbers of macrophages, proliferating macrophages and activated T-cells in the respective injured organs. The observation that these immune cells play key roles in driving end organ inflammation in multiple systemic rheumatic diseases have broad implications for the use of BTKB66 in managing patients with systemic rheumatic diseases where multiple end organs are afflicted, including lupus and systemic sclerosis.

Fatty Acid Amide Hydrolase Regulates Peripheral B Cell Receptor Revision, Polyreactivity, and B1 Cells in Lupus.

C57BL/6 mice bearing the Sle2(z) lupus-susceptibility congenic interval on chromosome 4 display high titers of polyclonal autoantibodies with generalized B cell hyperactivity, hallmarks of systemic lupus erythematosus. In B6.Sle2(z)HEL(Ig).sHEL BCR-transgenic mice, Sle2(z) did not breach central tolerance, but it led to heightened expression of endogenous Ig H and L chains in splenic B cells, upregulation of RAG, and serological polyreactivity, suggestive of excessive receptor revision. Fatty acid amide hydrolase (FAAH), a gene in the minimal subcongenic interval generated through recombinant mapping, was found to be upregulated in Sle2(z) B cells by microarray analysis, Western blot, and functional assays. Pharmacological inhibition of FAAH reversed the increase in receptor revision, RAG expression, and polyreactive autoantibodies in lupus-prone mice. These studies indicate that increased peripheral BCR revision, or selective peripheral expansion of BCR-revised B cells, may lead to systemic autoimmunity and that FAAH is a lupus-susceptibility gene that might regulate this process.

Prevention of murine lupus nephritis by targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid.

OBJECTIVE: Current treatment options for lupus are far from optimal. Previously, we reported that phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin, MEK-1/ERK-1,2, p38, STAT-3, STAT-5, NF-κB, multiple Bcl-2 family members, and various cell cycle molecules were overexpressed in splenic B cells in an age-dependent and gene dose-dependent manner in mouse strains with spontaneous lupus. Since the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) has been shown to inhibit AKT, MEK-1/2, and NF-κB, and to induce caspase-mediated apoptosis, we tested the therapeutic potential of this agent in murine lupus nephritis. METHODS: The synthetic triterpenoid CDDO-Me or placebo was administered to 2-month-old B6.Sle1.Sle3 mice or MRL/lpr mice, which develop spontaneous lupus. All mice were phenotyped for disease. RESULTS: CDDO-Me-treated mice exhibited significantly reduced splenic cellularity, with decreased numbers of both CD4+ T cells and activated CD69+/CD4+ T cells compared to the placebo-treated mice. These mice also exhibited a significant reduction in serum autoantibody levels, including anti-double-stranded DNA (anti-dsDNA) and antiglomerular antibodies. Finally, CDDO-Me treatment attenuated renal disease in mice, as indicated by reduced 24-hour proteinuria, blood urea nitrogen, and glomerulonephritis. At the mechanistic level, CDDO-Me treatment dampened MEK-1/2, ERK, and STAT-3 signaling within lymphocytes and oxidative stress. Importantly, the NF-E2-related factor 2 pathway was activated after CDDO-Me treatment, indicating that CDDO-Me may modulate renal damage in lupus via the inhibition of oxidative stress. CONCLUSION: These findings underscore the importance of AKT/MEK-1/2/NF-κB signaling in engendering murine lupus. Our findings indicate that the blockade of multiple signaling nodes and oxidative stress may effectively prevent and reverse the hematologic, autoimmune, and pathologic manifestations of lupus.

A role for IRF8 in B cell anergy.

B cell central tolerance is a process through which self-reactive B cells are removed from the B cell repertoire. Self-reactive B cells are generally removed by receptor editing in the bone marrow and by anergy induction in the periphery. IRF8 is a critical transcriptional regulator of immune system development and function. A recent study showed that marginal zone B cell and B1 B cell populations are dramatically increased in IRF8-deficient mice, indicating that there are B cell-developmental defects in the absence of IRF8. In this article, we report that mice deficient for IRF8 produced anti-dsDNA Abs. Using a hen egg lysozyme double-transgenic model, we further demonstrate that B cell anergy was breached in IRF8-deficient mice. Although anergic B cells in the IRF8-proficient background were blocked at the transitional stage of development, anergic B cells in the IRF8-deficient background were able to mature further, which allowed them to regain responses to Ag stimulation. Interestingly, our results show that IRF8-deficient B cells were more sensitive to Ag stimulation and were resistant to Ag-induced cell death. Moreover, our results show that IRF8 was expressed at a high level in the anergic B cells, and an elevated level of IRF8 promoted apoptosis in the transitional B cells. Thus, our findings reveal a previously unrecognized function of IRF8 in B cell anergy induction.

Cellular and molecular pathogenesis of systemic lupus erythematosus: lessons from animal models.

Systemic lupus erythematosus (SLE) is a complex disease characterized by the appearance of autoantibodies against nuclear antigens and the involvement of multiple organ systems, including the kidneys. The precise immunological events that trigger the onset of clinical manifestations of SLE are not yet well understood. However, research using various mouse strains of spontaneous and inducible lupus in the last two decades has provided insights into the role of the immune system in the pathogenesis of this disease. According to our present understanding, the immunological defects resulting in the development of SLE can be categorized into two phases: (a) systemic autoimmunity resulting in increased serum antinuclear and antiglomerular autoantibodies and (b) immunological events that occur within the target organ and result in end organ damage. Aberrations in the innate as well as adaptive arms of the immune system both play an important role in the genesis and progression of lupus. Here, we will review the present understanding--as garnered from studying mouse models--about the roles of various immune cells in lupus pathogenesis.

IRF4 is a suppressor of c-Myc induced B cell leukemia.

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator in B cell development and function. We have previously shown that IRF4, together with IRF8, orchestrates pre-B cell development by limiting pre-B cell expansion and by promoting pre-B cell differentiation. Here, we report that IRF4 suppresses c-Myc induced leukemia in EµMyc mice. Our results show that c-Myc induced leukemia was greatly accelerated in the IRF4 heterozygous mice (IRF4(+/-)Myc); the average age of mortality in the IRF4(+/-)Myc mice was only 7 to 8 weeks but was 20 weeks in the control mice. Our results show that IRF4(+/-)Myc leukemic cells were derived from large pre-B cells and were hyperproliferative and resistant to apoptosis. Further analysis revealed that the majority of IRF4(+/-)Myc leukemic cells inactivated the wild-type IRF4 allele and contained defects in Arf-p53 tumor suppressor pathway. p27(kip) is part of the molecular circuitry that controls pre-B cell expansion. Our results show that expression of p27(kip) was lost in the IRF4(+/-)Myc leukemic cells and reconstitution of IRF4 expression in those cells induced p27(kip) and inhibited their expansion. Thus, IRF4 functions as a classical tumor suppressor to inhibit c-Myc induced B cell leukemia in EµMyc mice.

Ikaros and Aiolos inhibit pre-B-cell proliferation by directly suppressing c-Myc expression.

Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell receptor and is dependent on cyclin D3 and c-Myc. We have shown previously that interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to suppress pre-B-cell proliferation. However, the molecular mechanisms through which Ikaros and Aiolos exert their growth inhibitory effect remain to be determined. Here, we provide evidence that Aiolos and Ikaros bind to the c-Myc promoter in vivo and directly suppress c-Myc expression in pre-B cells. We further show that downregulation of c-Myc is critical for the growth-inhibitory effect of Ikaros and Aiolos. Ikaros and Aiolos also induce expression of p27 and downregulate cyclin D3 in pre-B cells, and the growth-inhibitory effect of Ikaros and Aiolos is compromised in the absence of p27. A time course analysis further reveals that downregulation of c-Myc by Ikaros and Aiolos precedes p27 induction and cyclin D3 downregulation. Moreover, downregulation of c-Myc by Ikaros and Aiolos is necessary for the induction of p27 and downregulation of cyclin D3. Collectively, our studies identify a pre-B-cell receptor signaling induced inhibitory network, orchestrated by Ikaros and Aiolos, which functions to terminate pre-B-cell expansion.

A role for interferon regulatory factor 4 in receptor editing.

Receptor editing is the primary means through which B cells revise antigen receptors and maintain central tolerance. Previous studies have demonstrated that interferon regulatory factor 4 (IRF-4) and IRF-8 promote immunoglobulin light-chain rearrangement and transcription at the pre-B stage. Here, the roles of IRF-4 and -8 in receptor editing were analyzed. Our results show that secondary rearrangement was impaired in IRF-4 but not IRF-8 mutant mice, suggesting that receptor editing is defective in the absence of IRF-4. The role of IRF-4 in receptor editing was further examined in B-cell-receptor (BCR) transgenic mice. Our results show that secondary rearrangement triggered by membrane-bound antigen was defective in the IRF-4-deficient mice. Our results further reveal that the defect in secondary rearrangement is more severe at the immunoglobulin lambda locus than at the kappa locus, indicating that IRF-4 is more critical for the lambda rearrangement. We provide evidence demonstrating that the expression of IRF-4 in immature B cells is rapidly induced by self-antigen and that the reconstitution of IRF-4 expression in the IRF-4 mutant immature B cells promotes secondary rearrangement. Thus, our studies identify IRF-4 as a nuclear effector of a BCR signaling pathway that promotes secondary rearrangement at the immature B-cell stage.

Interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to down-regulate pre-B-cell receptor and promote cell-cycle withdrawal in pre-B-cell development.

Pre-B lymphocytes consist of 2 distinct cell populations: large pre-B and small pre-B. The large pre-B cells are newly generated pre-B cells that express pre-B-cell receptor (pre-BCR) on the surface and are highly proliferative; small pre-B cells are derived from large pre-B cells that have down-regulated pre-BCR and withdrawn from cell cycle. The molecular events that mediate the transition from cycling pre-B to small, resting pre-B have not been fully elucidated. Here, we show that interferon regulatory factors 4 and 8 (IRF4,8) suppress surrogate light chain expression and down-regulate pre-BCR in pre-B cells. Our studies further reveal that IRF4,8 induce the expression of Ikaros and Aiolos in pre-B cells, and reconstitution of expression of either one is sufficient to suppress surrogate light chain expression and down-regulate pre-BCR in pre-B cells lacking IRF4,8. Interestingly, our results also indicate that pre-B cells undergo growth inhibition and cell-cycle arrest in the presence of IRF4,8. Moreover, we provide evidence that Ikaros and Aiolos are indispensable for the down-regulation of pre-BCR and the cell-cycle withdrawal mediated by IRF4,8. Thus, IRF4,8 orchestrate the transition from large pre-B to small pre-B cells by inducing the expression of Ikaros and Aiolos.

Expressed sequence tags from feline uterine library.

Partial nucleotide sequences of 634 cDNAs randomly isolated from a feline uterine cDNA library (Stratagene) were determined by single pass sequencing. Homology search of the sequences to the non-redundant nucleotide databases revealed that 83% of the cDNAs matched registered feline or non-feline genes. Based on the gene identifications, these genes were predicted to be related with immunological, biochemical and regulatory functions in cats. Interestingly, the rest 17% of the cDNAs did not show homology to gene or EST sequence present in the nucleotide and protein databases, suggesting that these cDNAs include novel genes expressed only in the Felidae. This large scale sequencing of uterine cDNA will provide a useful molecular source for research not only towards health and disease conditions in cats but also in different fields of science where genetic information from cats will be of interest.