Angelina I. Bernardi

The pre-B cell receptor checkpoint

B lymphocytes are essential antibody‐producing cells of the immune system. During the development of progenitor B cells to mature B cells that express a membrane‐bound antibody, the B cell receptor (BCR), the cells undergo selection at several checkpoints, which ensures that a diverse antibody repertoire is generated and that the BCRs recognise foreign‐, but not self‐, antigens. In this review, we consider the pre‐BCR checkpoint. Mutations or alterations that affect this checkpoint underpin the development of pre‐B cell leukemias, primary immunodeficiency, and possibly, systemic autoimmunity.

Effects of lasofoxifene and bazedoxifene on B cell development and function.

The third generation selective estrogen receptor modulators lasofoxifene (las) and bazedoxifene (bza) are indicated for treatment of postmenopausal osteoporosis. 17β‐Estradiol (E2) and the second generation SERM raloxifene (ral) have major effects on the immune system, particularly on B cells. Treatment with E2 or ral inhibits B lymphopoiesis and treatment with E2, but not ral, stimulates antibody production. The effects of las and bza on the immune system have not been studied. Therefore, the aim of this study was to investigate their role in B cell development, maturation, and function. C57BL/6 mice were sham‐operated or ovariectomized (ovx) and treated with vehicle, E2, ral, las, or bza. All substances increased total bone mineral density in ovx mice, as measured by peripheral quantitative computed tomography. In uterus, bza alone lacked agonistic effect in ovx mice and even acted as an antagonist in sham mice. As expected, E2 decreased B cell numbers at all developmental stages from pre‐BI cells (in bone marrow) to transitional 1 (T1) B cells (in spleen) and increased marginal zone (MZ) B cells as determined by flow cytometry. However, treatment with las or bza only decreased the last stages of bone marrow B cell development and splenic T1 B cells, but had no effect MZ B cells. E2 increased antibody‐producing cells quantified by ELISPOT, but las or bza did not. In conclusion, las and bza differ from E2 by retaining normal number of cells at most B cell stages during B lymphopoiesis and maturation and by not increasing antibody‐producing cells.

Selective oestrogen receptor modulators lasofoxifene and bazedoxifene inhibit joint inflammation and osteoporosis in ovariectomised mice with collagen-induced arthritis

Objective. RA predominantly affects post-menopausal women and is strongly associated with development of generalised osteoporosis. To find treatments that target both joint manifestations and osteoporosis in RA is desirable. The third generation of selective oestrogen receptor modulators (SERMs) [lasofoxifene (LAS) and bazedoxifene (BZA)] are new treatment options for post-menopausal osteoporosis. The aim of this study was to investigate the effects of LAS and BZA on arthritic disease and inflammation-associated bone loss using CIA in mice. Methods. Female DBA/1 mice were ovariectomised and subjected to CIA as a model of post-menopausal RA. Mice received treatment with LAS, BZA, 17β-estradiol (E2) as reference or vehicle. Arthritis development was assessed and BMD was determined by peripheral quantitative CT of the femurs. Serologic markers of inflammation and cartilage destruction were analysed. Immune cells in lymph nodes were studied by flow cytometry. Results. LAS and BZA reduced the clinical severity of arthritis as well as the grade of histologic synovitis and erosions on cartilage and bone. Moreover, SERMs protected against generalised bone loss in CIA by increasing trabecular BMD. Both SERMs decreased serum marker of cartilage destruction and LAS reduced serum IL-6 levels. SERMs did not alter Th17 cells in lymph nodes as E2 did. Conclusion. The anti-osteoporotic drugs LAS and BZA were found to be potent inhibitors of joint inflammation and bone destruction in experimental arthritis. This study provides new important knowledge regarding the treatment regimen of post-menopausal women with RA who suffer from increased risk for osteoporosis.

Selective estrogen receptor modulators in T cell development and T cell dependent inflammation.

Lasofoxifene (las) and bazedoxifene (bza) are third generation selective estrogen receptor modulators (SERMs) with minimal estrogenic side effects, approved for treatment of postmenopausal osteoporosis. T cells are involved in the pathology of postmenopausal osteoporosis and previous studies have established an important role for 17β-estradiol (E2) in T cell development and function. E2 causes a drastic thymic atrophy, alters the composition of thymic T cell populations, and inhibits T cell dependent inflammation. In contrast, the second generation SERM raloxifene (ral) lacks these properties. Although las and bza are drugs approved for treatment of postmenopausal bone loss, it is of importance to study their effects on other biological aspects in order to extend the potential use of these compounds. Therefore, the aim of this study was to investigate if treatment with las and bza affects T lymphopoiesis and T cell dependent inflammation. C57Bl6 mice were ovariectomized (ovx) and treated with vehicle, E2, ral, las or bza. As expected, E2 reduced both thymus weight and decreased the proportion of early T cell progenitors while increasing more mature T cell populations in the thymus. E2 also suppressed the T cell dependent delayed-type hypersensitivity (DTH) reaction to oxazolone (OXA). Ral and las, but not bza, decreased thymus weight, while none of the SERMs had any effects on T cell populations in the thymus or on inflammation in DTH. In conclusion, this study shows that treatment with las or bza does not affect T lymphopoiesis or T cell dependent inflammation.

Absence of surrogate light chain results in spontaneous autoreactive germinal centres expanding V H 81X -expressing B cells

Random recombination of antibody heavy- and light-chain genes results in a diverse B-cell receptor (BCR) repertoire including self-reactive BCRs. However, tolerance mechanisms that prevent the development of self-reactive B cells remain incompletely understood. The absence of the surrogate light chain, which assembles with antibody heavy chain forming a pre-BCR, leads to production of antinuclear antibodies (ANAs). Here we show that the naive follicular B-cell pool is enriched for cells expressing prototypic ANA heavy chains in these mice in a non-autoimmune background with a broad antibody repertoire. This results in the spontaneous formation of T-cell-dependent germinal centres that are enriched with B cells expressing prototypic ANA heavy chains. However, peripheral tolerance appears maintained by selection thresholds on cells entering the memory B-cell and plasma cell pools, as exemplified by the exclusion of cells expressing the intrinsically self-reactive V(H)81X from both pools.

Suppression of Experimental Arthritis and Associated Bone Loss by a Tissue-Selective Estrogen Complex

In addition to the systemic inflammation present in rheumatoid arthritis (RA), decreased estradiol levels in postmenopausal RA patients further accelerate bone loss in these patients. The tissue-selective estrogen complex (TSEC), an estrogen combined with a selective estrogen receptor modulator, is a new hormone replacement therapy option. The first approved TSEC, containing conjugated estrogens and bazedoxifene (BZA), reduces menopausal symptoms and prevents osteoporosis with an improved safety profile compared with conventional hormone replacement therapy. Previous studies have shown that estrogens strongly inhibit experimental arthritis whereas BZA is mildly suppressive. In this study the antiarthritic potential of combined BZA and estradiol is explored for the first time. Female ovariectomized DBA/1 mice were subjected to collagen-induced arthritis, an experimental postmenopausal RA model, and treated with BZA, 17β-estradiol (E2), combined BZA and E2 (BZA/E2), or vehicle. BZA/E2 suppressed arthritis severity and frequency, synovitis, and joint destruction, equally efficient as E2 alone. Unwanted estrogenic proliferative effects on the endometrium were blocked by the addition of BZA, determined by collecting uterine weights. Bone mineral density was measured by peripheral quantitative computed tomography, and all treatments protected collagen-induced arthritis mice from both trabecular and cortical bone loss. Moreover, BZA/E2, but not E2 alone, inhibited preosteoclast formation and reduced serum anticollagen type II antibodies. In conclusion, a TSEC, herein combined BZA/E2, suppresses experimental arthritis and prevents associated bone loss as efficiently as E2 alone but with minimal uterine effects, highlighting the need for clinical trials that evaluate the addition of a TSEC to conventional postmenopausal RA treatment.

α7 Nicotinic Acetylcholine Receptor Is Expressed in Human Atherosclerosis and Inhibits Disease in Mice

Objective— Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the &agr;7 nicotinic acetylcholine receptor (&agr;7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr –/– mouse model. Approach and Results— &agr;7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of &agr;7nAChR signaling in atherosclerosis, male Ldlr –/– mice were lethally irradiated and reconstituted with bone marrow from wild-type or &agr;7nAChR-deficient animals. Ablation of hematopoietic cell &agr;7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-&ggr; mRNA, implying increased Th1 activity in the absence of &agr;7nAChR signaling. Conclusions— The present study shows that signaling through hematopoietic &agr;7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that &agr;7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.

Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells

Selection of the primary antibody repertoire takes place in pro‐/pre‐B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B‐cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody‐producing cells is due to an inability of the resultant B‐cell receptors to induce central and/or peripheral B‐cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B‐cell pool. Those that enter the marginal zone (MZ) B‐cell pool escape selection and survive, possibly due to the B‐lymphopenia and elevated levels of B‐cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti‐dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B‐cell tolerance and inhibits anti‐DNA antibody production by MZ B cells.

Roles of activating functions 1 and 2 of estrogen receptor α in lymphopoiesis

Apart from the role of sex steroids in reproduction, sex steroids are also important regulators of the immune system. 17β-estradiol (E2) represses T and B cell development, but augments B cell function, possibly explaining the different nature of immune responses in men and women. Both E2 and selective estrogen receptors modulators (SERM) act via estrogen receptors (ER). Activating functions (AF)-1 and 2 of the ER bind to coregulators and thus influence target gene transcription and subsequent cellular response to ER activation. The importance of ERαAF-1 and AF-2 in the immunomodulatory effects of E2/SERM has previously not been reported. Thus, detailed studies of T and B lymphopoiesis were performed in ovariectomized E2-, lasofoxifene- or raloxifene-treated mice lacking either AF-1 or AF-2 domains of ERα, and their wild-type littermate controls. Immune cell phenotypes were analyzed with flow cytometry. All E2 and SERM-mediated inhibitory effects on thymus cellularity and thymic T cell development were clearly dependent on both ERαAFs. Interestingly, divergent roles of ERαAF-1 and ERαAF-2 in E2 and SERM-mediated modulation of bone marrow B lymphopoiesis were found. In contrast to E2, effects of lasofoxifene on early B cells did not require functional ERαAF-2, while ERαAF-1 was indispensable. Raloxifene reduced early B cells partly independent of both ERαAF-1 and ERαAF-2. Results from this study increase the understanding of the impact of ER modulation on the immune system, which can be useful in the clarification of the molecular actions of SERMs and in the development of new SERM.

Effects of a tissue-selective estrogen complex on B lymphopoiesis and B cell function

BACKGROUND/OBJECTIVE 17β-estradiol (E2) has major effects on the immune system. It induces thymic atrophy, inhibits both T and B lymphopoiesis and stimulates antibody production treatment with E2 has protective effects on the skeleton but is associated with negative side effects in reproductive organs. A tissue-selective estrogen complex (TSEC) comprise of estrogens combined with a selective estrogen receptor modulator (SERM). TSEC therapy displays the bone-protective effects of estrogen, while the negative side effects on reproductive organs are blocked by the SERM. In a recent publication we showed that treatment with the TSEC E2+bazedoxifene (bza) potently inhibits experimental arthritis and associated osteoporosis. In order to elucidate immunological mechanisms involved in those effects, the aim of this study was to investigate how E2+bza treatment affects the healthy immune system. METHODS Ovariectomized C57BL/6N mice were treated with vehicle, E2, bza or E2+bza. Weights of uterus and thymus were determined and fluorescence-activated cell sorting was used to analyze B cell populations in bone marrow and spleen. Immunoglobulin production from B cells in bone marrow and spleen were determined using ELISPOT. RESULTS Addition of bza to E2-treatment totally antagonized the E2-mediated proliferative effect on uterus. On the contrary, addition of bza to E2-treatment did not block the E2-induced thymic atrophy or inhibition of B lymphopoiesis, and did not block the E2-induced increase in immunoglobulin secretion from bone marrow B cells. CONCLUSIONS Addition of bza to E2-treatment blocks E2-induced uteroproliferation but does not alter E2-mediated effects on thymus, B lymphopoiesis or B cell function.