Role of estrogen signaling in fibroblastic reticular cells for innate and adaptive immune responses in antigen-induced arthritis.

Women are more prone to develop rheumatoid arthritis, with peak incidence occurring around menopause. Estrogen has major effects on the immune system and is protective against arthritis. We have previously shown that treatment with estrogen inhibits inflammation and joint destruction in murine models of arthritis, although the mechanisms involved remain unclear. Fibroblastic reticular cells (FRCs) are specialized stromal cells that generate the three-dimensional structure of lymph nodes (LNs). FRCs are vital for coordinating immune responses from within LNs and are characterized by the expression of the chemokine CCL19, which attracts immune cells. The aim of this study was to determine whether the influence of estrogen on innate and adaptive immune cells in arthritis is mediated by estrogen signaling in FRCs. Conditional knockout mice lacking estrogen receptor α (ERα) in CCL19-expressing cells (Ccl19-CreERαfl/fl) were generated and tested. Ccl19-CreERαfl/fl mice and littermate controls were ovariectomized, treated with vehicle or estradiol and subjected to the 28-day-long antigen-induced arthritis model to enable analyses of differentiated T- and B-cell populations and innate cells in LNs by flow cytometry. The results reveal that while the response to estradiol treatment in numbers of FRCs per LN is significantly reduced in mice lacking ERα in FRCs, estrogen does not inhibit joint inflammation or markedly affect immune responses in this arthritis model. Thus, this study validates the Ccl19-CreERαfl/fl strain for studying estrogen signaling in FRCs within inflammatory diseases, although the chosen arthritis model is deemed unsuitable for addressing this question.

The Pseudomonas aeruginosa lectin LecB modulates intracellular reactive oxygen species production in human neutrophils.

Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen ubiquitously present throughout nature. LecB, a fucose-, and mannose-binding lectin, is a prominent virulence factor of P. aeruginosa, which can be expressed on the bacterial surface but also be secreted. However, the LecB interaction with human immune cells remains to be characterized. Neutrophils comprise the first line of defense against infections and their production of reactive oxygen species (ROS) and release of extracellular traps (NETs) are critical antimicrobial mechanisms. When profiling the neutrophil glycome we found several glycoconjugates on granule and plasma membranes that could potentially act as LecB receptors. In line with this, we here show that soluble LecB can activate primed neutrophils to produce high levels of intracellular ROS (icROS), an effect that was inhibited by methyl fucoside. On the other hand, soluble LecB inhibits P. aeruginosa-induced icROS production. In support of that, during phagocytosis of wild-type and LecB-deficient P. aeruginosa, bacteria with LecB induced less icROS production as compared with bacteria lacking the lectin. Hence, LecB can either induce or inhibit icROS production in neutrophils depending on the circumstances, demonstrating a novel and potential role for LecB as an immunomodulator of neutrophil functional responses.

Transplantation of gut microbiota from old mice into young healthy mice reduces lean mass but not bone mass.

Aging is associated with low bone and lean mass as well as alterations in the gut microbiota (GM). In this study, we determined whether the reduced bone mass and relative lean mass observed in old mice could be transferred to healthy young mice by GM transplantation (GMT). GM from old (21-month-old) and young adult (5-month-old) donors was used to colonize germ-free (GF) mice in three separate studies involving still growing 5- or 11-week-old recipients and 17-week-old recipients with minimal bone growth. The GM of the recipient mice was similar to that of the donors, demonstrating successful GMT. GM from old mice did not have statistically significant effects on bone mass or bone strength, but significantly reduced the lean mass percentage of still growing recipient mice when compared with recipients of GM from young adult mice. The levels of propionate in the cecum of mice receiving old donor GM were significantly lower than those in mice receiving young adult donor GM. Bacteroides ovatus was enriched in the microbiota of recipient mice harboring GM from young adult donors. The presence of B. ovatus was not only significantly associated with high lean mass percentage in mice, but also with lean mass adjusted for fat mass in the large human HUNT cohort. In conclusion, GM from old mice reduces lean mass percentage but not bone mass in young, healthy, still growing recipient mice. Future studies are warranted to determine whether GM from young mice improves the musculoskeletal phenotype of frail elderly recipient mice.

Profiling of innate and adaptive immune cells during influenza virus infection reveals sex bias in invariant natural killer T (iNKT) cells.

BACKGROUND: Influenza A virus (IAV) infection leads to significant morbidity and mortality. Biological sex influences the immune responses to IAV infection, resulting in higher mortality in women of reproductive age. Previous studies revealed increased activation of T and B cells in female mice after IAV infection, but extensive analysis of sex differences in both innate and adaptive immune cells over time is lacking. Invariant natural killer T (iNKT) cells are fast-reacting forces and modulators of immune responses that are important to IAV immunity, but it is not known if the presence and function of iNKT cells differ between females and males. The aim of this study was to determine immunological mechanisms that contribute to the increased disease severity in female mice during IAV infection. METHODS: Female and male mice were infected with mouse-adapted IAV and monitored for weight loss and survival. Immune cell populations and cytokine expression in bronchoalveolar lavage fluid, lung, and mediastinal lymph node were determined at three time points after infection using flow cytometry and ELISA. RESULTS: The results reveal increased severity and mortality in adult female mice compared to age-matched males. Female mice show larger increases in innate and adaptive immune cell populations and cytokine production in lung compared to mock on Day 6 postinfection. On Day 9 postinfection, female mice express higher numbers of iNKT cells in lung and liver compared to males. CONCLUSIONS: This comprehensive analysis of immune cells and cytokines over time following IAV infection reveals increased leukocyte expansion and stronger proinflammatory cytokine responses in female mice during disease initiation. Furthermore, this is the first study to report a sex bias in iNKT cell populations after IAV infection. The data suggests that the process of recovery from IAV-induced airway inflammation is associated with increased expansion of several different iNKT cell subpopulations in female mice.

Physiological estrogen levels are dispensable for the sex difference in immune responses during allergen-induced airway inflammation.

Women show an increased prevalence of adult-onset asthma compared to men and previous studies have shown that testosterone inhibits while estrogen worsens allergen-induced airway inflammation. However, detailed knowledge about the aggravating effects of estrogen on immune responses remain unclear. Defining the effects of physiological levels of estrogen on immune responses in asthma would aid in the development of improved treatment strategies. In this study, the importance of estrogen for the sex difference in asthma was determined using a murine model of house dust mite (HDM)-induced airway inflammation on intact female and male mice, as well as on ovariectomized (OVX) female mice treated with a physiological dose of 17ß-estradiol (E2). Innate and adaptive immune responses were defined in bronchoalveolar lavage fluid, mediastinal lymph node (mLN) and lung tissue. The results reveal increased numbers of lung eosinophils, macrophages, and dendritic cells in female but not in male mice after HDM challenge. Females also exhibit higher numbers of Th17 cells in both mLN and lung in response to HDM. However, treatment of OVX mice with physiological levels of E2 does not influence any of the analyzed cell populations. Together, this study confirms the previously reported sex difference in allergen-induced airway inflammation and show that female mice mount stronger innate and adaptive immune responses to HDM challenge, but these effects are not mediated by physiological levels of E2.

Reduction of Mature B Cells and Immunoglobulins Results in Increased Trabecular Bone.

Inflammation has a significant effect on bone remodeling and can result in bone loss via increased stimulation of osteoclasts. Activated immunoglobulins, especially autoantibodies, can increase osteoclastogenesis and are associated with pathological bone loss. Whether immunoglobulins and mature B lymphocytes are important for general bone architecture has not been completely determined. Here we demonstrate, using a transgenic mouse model, that reduction of mature B cells and immunoglobulins leads to increased trabecular bone mass compared to wild-type (WT) littermate controls. This bone effect is associated with a decrease in the number of osteoclasts and reduced bone resorption, despite decreased expression of osteoprotegerin. We also demonstrate that the reduction of mature B cells and immunoglobulins do not prevent bone loss caused by estrogen deficiency or arthritis compared to WT littermate controls. In conclusion, the reduction of mature B cells and immunoglobulins results in disturbed regulation of trabecular bone turnover in healthy conditions but is dispensable for pathological bone loss. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ERα Signaling in a Subset of CXCL12-Abundant Reticular Cells Regulates Trabecular Bone in Mice.

Estrogen has pronounced effects on the immune system, which also influences bone homeostasis. In recent years, stromal cells in lymphoid organs have gained increasing attention as they not only support the regulation of immune responses but also affect bone remodeling. A conditional knockout mouse model where estrogen receptor alpha (ERα) is deleted in CCL19-expressing stromal cells (Ccl19-Cre ERα fl/fl mice) was generated and bone densitometry was performed to analyze the importance of stromal cell-specific ERα signaling on the skeleton. Results showed that female Ccl19-Cre ERα fl/fl mice display reduced total bone mineral density and detailed X-ray analyses revealed that ERα expression in CCL19-expressing stromal cells is important for trabecular but not cortical bone homeostasis. Further analysis showed that the trabecular bone loss is caused by increased osteoclastogenesis. Additionally, the bone formation rate was reduced; however, the expression of osteoprogenitor genes was not altered. Analysis of the bone marrow stromal cell compartment revealed a deletion of ERα in a subgroup of CXCL12-abundant reticular (CAR) cells resulting in increased secretion of the pro-osteoclastogenic chemokine CXCL12. In conclusion, this study reveals the importance of ERα signaling in CAR cells for bone health. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Physiological levels of estradiol limit murine osteoarthritis progression.

Among patients with knee osteoarthritis (OA), postmenopausal women are over-represented. The purpose of this study was to determine whether deficiency of female sex steroids affects OA progression and to evaluate the protective effect of treatment with a physiological dose of 17ß-estradiol (E2) on OA progression using a murine model. Ovariectomy (OVX) of female mice was used to mimic a postmenopausal state. OVX or sham-operated mice underwent surgery for destabilization of the medial meniscus (DMM) to induce OA. E2 was administered in a pulsed manner for 2 and 8 weeks. OVX of OA mice did not influence the cartilage phenotype or synovial thickness, while both cortical and trabecular subchondral bone mineral density (BMD) decreased after OVX compared with sham-operated mice at 8 weeks post-DMM surgery. Additionally, OVX mice displayed decreased motor activity, reduced threshold of pain sensitivity, and increased number of T cells in the inguinal lymph nodes compared to sham-operated mice 2 weeks after OA induction. Eight weeks of treatment with E2 prevented cartilage damage and thickening of the synovium in OVX OA mice. The motor activity was improved after E2 replacement at the 2 weeks time point, which was also associated with lower pain sensitivity in the OA paw. E2 treatment protected against OVX-induced loss of subchondral trabecular bone. The number of T cells in the inguinal lymph nodes was reduced by E2 treatment after 8 weeks. This study demonstrates that treatment with a physiological dose of E2 exerts a protective role by reducing OA symptoms.

A tissue-specific role of membrane-initiated ERα signaling for the effects of SERMs.

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ER) agonists or antagonists in a tissue-specific manner. ERs exert effects via nuclear actions but can also utilize membrane-initiated signaling pathways. To determine if membrane-initiated ERα (mERα) signaling affects SERM action in a tissue-specific manner, C451A mice, lacking mERα signaling due to a mutation at palmitoylation site C451, were treated with Lasofoxifene (Las), Bazedoxifene (Bza), or estradiol (E2), and various tissues were evaluated. Las and Bza treatment increased uterine weight to a similar extent in C451A and control mice, demonstrating mERα-independent uterine SERM effects, while the E2 effect on the uterus was predominantly mERα-dependent. Las and Bza treatment increased both trabecular and cortical bone mass in controls to a similar degree as E2, while both SERM and E2 treatment effects were absent in C451A mice. This demonstrates that SERM effects, similar to E2 effects, in the skeleton are mERα-dependent. Both Las and E2 treatment decreased thymus weight in controls, while neither treatment affected the thymus in C451A mice, demonstrating mERα-dependent SERM and E2 effects in this tissue. Interestingly, both SERM and E2 treatments decreased the total body fat percent in C451A mice, demonstrating the ability of these treatments to affect fat tissue in the absence of functional mERα signaling. In conclusion, mERα signaling can modulate SERM responses in a tissue-specific manner. This novel knowledge increases the understanding of the mechanisms behind SERM effects and may thereby facilitate the development of new improved SERMs.

A tissue-selective estrogen complex as treatment of osteoporosis in experimental lupus.

Osteoporosis is a common secondary complication in patients with systemic lupus erythematosus (SLE). Current osteoporosis treatment with bisphosphonates has some negative side effects and there is a lack of data regarding newer treatments options for SLE associated osteoporosis. The tissue-selective estrogen complex (TSEC) containing conjugated estrogens and the selective estrogen receptor modulator bazedoxifene (Bza) is approved for treatment of postmenopausal vasomotor symptoms and prevention of osteoporosis. However, it has not been evaluated for treatment of osteoporosis in postmenopausal SLE patients. Ovariectomized MRL/lpr mice constitute a model for postmenopausal lupus that can be used for osteoporosis studies. We used this model in a set of experiments where the mice were treated with different doses of 17ß-estradiol-3-benzoate (E2), Bza, or TSEC (E2 plus Bza), administered in the early or late phases of disease development. The skeleton was analyzed by dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, and high-resolution microcomputed tomography. The lupus disease was assessed by determination of proteinuria, hematuria, and lupus disease markers in serum. Treatment with medium dose TSEC administered in early disease protected ovariectomized MRL/lpr mice from trabecular bone loss, while there were no differences in lupus disease parameters between treatments. This is the first experimental study to investigate TSEC as a potential new therapy for osteoporosis in postmenopausal SLE.

Pulsed administration for physiological estrogen replacement in mice.

Estrogens are important regulators of body physiology and have major effects on metabolism, bone, the immune- and central nervous systems. The specific mechanisms underlying the effects of estrogens on various cells, tissues and organs are unclear and mouse models constitute a powerful experimental tool to define the physiological and pathological properties of estrogens. Menopause can be mimicked in animal models by surgical removal of the ovaries and replacement therapy with 17ß-estradiol in ovariectomized (OVX) mice is a common technique used to determine specific effects of the hormone. However, these studies are complicated by the non-monotonic dose-response of estradiol, when given as therapy. Increased knowledge of how to distribute estradiol in terms of solvent, dose, and administration frequency, is required in order to accurately mimic physiological conditions in studies where estradiol treatment is performed. In this study, mice were OVX and treated with physiological doses of 17ß-estradiol-3-benzoate (E2) dissolved in miglyol or PBS. Subcutaneous injections were performed every 4 days to resemble the estrus cycle in mice. Results show that OVX induces an osteoporotic phenotype, fat accumulation and impairment of the locomotor ability, as expected. Pulsed administration of physiological doses of E2 dissolved in miglyol rescues the phenotypes induced by OVX. However, when E2 is dissolved in PBS the effects are less pronounced, possibly due to rapid wash out of the steroid.

Intermediate monocytes correlate with CXCR3+ Th17 cells but not with bone characteristics in untreated early rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with development of generalized osteoporosis. Bone-degrading osteoclasts are derived from circulating precursor cells of monocytic lineage, and the intermediate monocyte population is important as osteoclast precursors in inflammatory conditions. T cells of various subsets are critical in the pathogenesis of both RA and associated osteoporosis, but so far, no studies have examined associations between circulating intermediate monocytes, T cell subsets and bone characteristics in patients with RA. The aim of this study was to investigate the frequency of intermediate monocytes in patients with untreated early rheumatoid arthritis (ueRA) compared to healthy controls (HC), and to explore the correlation between intermediate monocytes and a comprehensive panel of T helper cell subsets, bone density and bone microarchitecture in ueRA patients. METHODS: 78 patients with ueRA fulfilling the ACR/EULAR 2010 criteria were included and compared to 29 age- and sex-matched HC. Peripheral blood samples were obtained before start of treatment and proportions of monocyte subsets and CD4+ helper and regulatory T cell subsets were analyzed by flow cytometry. Bone densitometry was performed on 46 of the ueRA patients at inclusion using DXA and HR-pQCT. RESULTS: Flow cytometric analyses showed that the majority of ueRA patients had frequencies of intermediate monocytes comparable to HC. The intermediate monocyte population correlated positively with CXCR3+ Th17 cells in ueRA patients but not in HC. However, neither the proportions of intermediate monocytes nor CXCR3+ Th17 cells were associated with bone density or bone microarchitecture measurements. CONCLUSIONS: Our findings suggest that in early RA, the intermediate monocytes do not correlate with bone characteristics, despite positive correlation with circulating CXCR3+ Th17 cells. Future longitudinal studies in patients with longer disease duration are required to fully explore the potential of intermediate monocytes to drive bone loss in RA.

Pasteurized Akkermansia muciniphila protects from fat mass gain but not from bone loss.

Probiotic bacteria can protect from ovariectomy (ovx)-induced bone loss in mice. Akkermansia muciniphila is considered to have probiotic potential due to its beneficial effect on obesity and insulin resistance. The purpose of the present study was to determine if treatment with pasteurized Akkermansia muciniphila (pAkk) could prevent ovx-induced bone loss. Mice were treated with vehicle or pAkk for 4 wk, starting 3 days before ovx or sham surgery. Treatment with pAkk reduced fat mass accumulation confirming earlier findings. However, treatment with pAkk decreased trabecular and cortical bone mass in femur and vertebra of gonadal intact mice and did not protect from ovx-induced bone loss. Treatment with pAkk increased serum parathyroid hormone (PTH) levels and increased expression of the calcium transporter Trpv5 in kidney suggesting increased reabsorption of calcium in the kidneys. Serum amyloid A 3 (SAA3) can suppress bone formation and mediate the effects of PTH on bone resorption and bone loss in mice and treatment with pAkk increased serum levels of SAA3 and gene expression of Saa3 in colon. Moreover, regulatory T cells can be protective of bone and pAkk-treated mice had decreased number of regulatory T cells in mesenteric lymph nodes and bone marrow. In conclusion, treatment with pAkk protected from ovx-induced fat mass gain but not from bone loss and reduced bone mass in gonadal intact mice. Our findings with pAkk differ from some probiotics that have been shown to protect bone mass, demonstrating that not all prebiotic and probiotic factors have the same effect on bone.

The late endosomal adaptor molecule p14 (LAMTOR2) regulates TGFß1-mediated homeostasis of Langerhans cells.

Langerhans cells (LCs), a sub-population of dendritic cells (DCs) in the skin, participate in the regulation of immunity and peripheral tolerance. The adaptor molecule p14 is part of the late endosomal/lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin (mTOR) activator/regulator (LAMTOR) complex, which mediates the activation of lysosome-associated extracellular signaling-regulated kinase (ERK) and the mTOR cascade. In previous work, we demonstrated that CD11c-specific deficiency of p14 disrupts LC homeostasis by affecting the LAMTOR-mediated ERK and mTOR signaling. In this study, we extended our analysis on p14 deficiency specifically in LCs. Langerin-specific ablation of p14 caused a complete loss of LCs, accompanied by an increased maturational phenotype of LCs. The absence of LCs in p14-deficient mice reduced contact hypersensitivity (CHS) responses to the contact sensitizer trinitrochlorobenzene. Analysis using bone marrow-derived DCs (BMDCs) revealed that p14 deficiency in DCs/LCs interfered with the LC-relevant transforming growth factor ß1 (TGFß1) pathway, by lowering TGFß receptor II expression on BMDCs and LCs, as well as surface binding of TGFß1 on BMDCs. We conclude that p14 deficiency affects TGFß1 sensitivity of LCs, which is mandatory for their homeostasis and subsequently for their immunological function during CHS.

LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling.

The receptor tyrosine kinase Flt3 and its ligand are crucial for dendritic cell (DC) homeostasis by activating downstream effectors including mammalian target of Rapamycin (mTOR) signalling. LAMTOR2 is a member of the Ragulator/LAMTOR complex known to regulate mTOR and extracellular signal-regulated kinase activation on the late endosome as well as endosomal biogenesis. Here we show in mice that conditional ablation of LAMTOR2 in DCs results in a severe disturbance of the DC compartment caused by accumulation of Flt3 on the cell surface. This results in an increased downstream activation of the AKT/mTOR signalling pathway and subsequently to a massive expansion of conventional DCs and plasmacytoid DCs in ageing mice. Finally, we can revert the symptoms in vivo by inhibiting the activation of Flt3 and its downstream target mTOR.

The late endosomal p14-MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration.

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14-MP1 (LAMTOR2/3) complex in FA dynamics. p14-MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end-directed traffic of p14-MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14-MP1 scaffold complex to the vicinity of FAs.

Mild fixation and permeabilization protocol for preserving structures of endosomes, focal adhesions, and actin filaments during immunofluorescence analysis.

Intracellular membrane trafficking is a highly dynamic process to sort proteins into either the recycling or degradation pathway. The late endosome is a major component of this endosomal biogenesis toward degradation by the lysosome. The endocytotic system is spread throughout the cytoplasm, and vesicle motility is achieved by multiple proteins including Rabs, motor proteins, and cytostructural elements. The subcellular localization of the late endosome is distributed from the accumulation in the perinuclear region toward the cell periphery. Using immunofluorescence methods combined with live-cell microscopy, we want to show that the preservation of the peripheral late endosomal compartment can be successfully achieved by two different techniques. On one hand, we compare two different widely used permeabilization methods: Triton X-100 and saponin. Comparing live-cell microscopic pictures of the same cell with immunofluorescences after fixation and permeabilization revealed improved results by the use of saponin. On the other hand, we present here a protocol of mild fixation to preserve peripheral structures like focal adhesion in combination with endosomes and actin filaments.

The late endosomal adaptor molecule p14 (LAMTOR2) represents a novel regulator of Langerhans cell homeostasis.

Langerhans cells (LCs) are dendritic cells (DCs) residing in epithelia, where they critically regulate immunity and tolerance. The p14 adaptor molecule is part of the late endosomal/LAMTOR (lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin [mTOR] activator/regulator) complex, thereby contributing to the signal transduction of the extracellular signaling-regulated kinase (ERK) and the mTOR cascade. Furthermore, p14 represents an important regulator for endosomal sorting processes within the cell. Mutated, dysfunctional p14 leads to a human immunodeficiency disorder with endosomal/lysosomal defects in immune cells. Because p14 participates in the regulation of endosomal trafficking, growth factor signaling, and cell proliferation, we investigated the role of p14 in mouse DCs/LCs using a conditional knockout mouse model. p14-deficient animals displayed a virtually complete loss of LCs in the epidermis early after birth due to impaired proliferation and increased apoptosis of LCs. Repopulation analysis after application of contact sensitizer leads to the recruitment of a transient LC population, predominantly consisting of short-term LCs. The underlying molecular mechanism involves the p14-mediated disruption of the LAMTOR complex which results in the malfunction of both ERK and mTOR signal pathways. Hence, we conclude that p14 acts as a novel and essential regulator of LC homeostasis in vivo.