Faculty Bibliography

OBJECTIVE:The protagonism of regulatory B cells seems to vary along the course of the disease in murine models of inflammatory conditions. Decreased numbers of circulating regulatory CD19+CD24hiCD38hi transitional (cTr) B cells have been described in patients with long-standing RA, thus our objective was to examine the frequency and evolution of cTr B cells in the peripheral blood of early RA (ERA) patients. METHODS:Freshly isolated peripheral blood mononuclear cells from 48 steroid- and DMARD-naïve ERA patients with a disease duration of <24 weeks and 48 healthy controls (HCs) were examined by flow cytometry. Co-cultures of isolated memory B cells were established with autologous T cells in the absence or presence of Tr B cells. RESULTS:As compared with HCs, ERA patients demonstrated an increased frequency of cTr B cells. cTr B cells of ERA patients and HCs displayed an anti-inflammatory cytokine profile and were able to downregulate T cell IFN-γ and IL-21 production, together with ACPA secretion in autologous B/T cell co-cultures. Basal frequencies of cTr B cells above the median value observed in HCs were associated with a good EULAR response to MTX at 12 months [relative risk 2.91 (95% CI 1.37, 6.47)]. A significant reduction of cTr B cells was observed 12 months after initiating MTX, when the cTr B cell frequency was no longer elevated but decreased, and this was independent of the degree of clinical response or the intake of prednisone. CONCLUSION/CONCLUSIONS:An increased frequency of regulatory cTr B cells is apparent in untreated ERA and the baseline cTr B cell frequency is associated with the clinical response to MTX at 12 months.

OBJECTIVES/OBJECTIVE:To describe the frequency and causes for the presence of a halo sign on the ultrasound of patients without a diagnosis of GCA. METHODS:In total, 305 patients with temporal artery colour Doppler ultrasound showing the presence of halo sign (intima-media thickness ≥0.34 mm for temporal arteries [TAs] and ≥1 mm for axillary arteries) were included, and their medical records were reviewed. The clinical diagnosis based on the evolution of the patient over at least one year was established as the definitive diagnosis. RESULTS:Fourteen of the 305 (4.6%) patients included showed presence of the halo sign without final diagnosis of GCA: 12 patients in the TAs (86%), and two patients with isolated AAs involvement (14%). Their diagnoses were PMR (n = 4, 29%); atherosclerosis (n = 3, 21%); and non-Hodgkin lymphoma type T, osteomyelitis of the skull base, primary amyloidosis associated with multiple myeloma, granulomatosis with polyangiitis, neurosyphilis, urinary sepsis and narrow-angle glaucoma (n = 1 each, 7%). CONCLUSION/CONCLUSIONS:The percentage of halo signs on the ultrasound of patients without GCA is low, but it does exist. There are conditions that may also show the halo sign (true positive halo sign), and we must know these and always correlate the ultrasound findings with the patient's clinic records.

OBJECTIVE:A novel population of B helper cells, phenotypically CD4+CXCR5-PD-1hi, has been described in the synovial tissues and peripheral blood of seropositive RA patients, and termed 'peripheral helper T' (Tph) cells. Contrary to CD4+CXCR5+PD-1hi follicular helper T (Tfh), Tph cells are not located in lymphoid organs but accumulate in inflamed tissues. Our objective was to study the frequency of circulating Tph (cTph) and circulating Tfh cell counterparts (cTfh) in patients with early RA (eRA). METHODS:Freshly isolated peripheral blood mononuclear cells from 56 DMARD-naïve eRA patients and 56 healthy controls were examined by flow cytometry. Autologous cocultures of naïve or memory B cells were established with isolated peripheral blood Tph or Tfh cells. RESULTS:Seropositive (RF+ and/or ACPA+, n = 38) but not seronegative eRA patients (n = 18) demonstrated increased frequencies and absolute numbers of cTph and cTfh cells. cTph but not cTfh cells expressed CCR2. Those eRA patients who experienced a significant clinical improvement at 12 months demonstrated a marked decrease of their cTph cell numbers whereas their cTfh cell numbers remained unchanged. Both isolated Tph and isolated Tfh cells were able to induce maturation of memory B cells, whereas only Tfh cells could differentiate naïve B cells. CONCLUSION/CONCLUSIONS:Two populations of PD-1hiCD4 T cells with distinct phenotype and B cell helping capacity are increased in the peripheral blood of seropositive eRA patients. Whereas cTph cells are present only in patients with an active disease, cTfh cells seem to be constitutively elevated.

Our objective was to study the frequency of circulating CD19+CD24hiCD38hi B cells (Breg) in AS patients. To this end, peripheral blood was drawn from AS patients naïve for TNF blockers (AS/nb) (n = 42) and healthy controls (HC) (n = 42). Six patients donated blood for a second time, 6 months after initiating treatment with anti-TNFα drugs. After isolation by Ficoll-Hypaque, PBMCs were stained with antibodies to CD3, CD4, CD19, CD24, and CD38, and examined by cytometry. For functional studies, total CD19+ B cells were isolated from PBMCs of 3 HC by magnetical sorting. Breg-depleted CD19+ B cells were obtained after CD19+CD24hiCD38hi B cells were removed from total CD19+ cells by cytometry. Total CD19+ B cells or Breg-depleted CD19+ B cells were established in culture and stimulated through their BCR. Secretion of IFNγ was determined by ELISA in culture supernatants. When compared with HC, AS/nb patients demonstrated a significantly increased frequency of Breg cells, which was independent of disease activity. Anti-TNFα drugs induced a significant reduction of circulating Breg numbers, which were no longer elevated after six months of treatment. Functional in vitro studies showed that the secretion of IFNγ was significantly higher in Breg-depleted as compared with total CD19+ B cells, indicating that Breg can downmodulate B cell pro-inflammatory cytokine secretion. In summary, an increased frequency of circulating CD19+CD24hiCD38hi B cells is observed in AS/nb patients, that is not related with disease activity; anti-TNFα drugs are able to downmodulate circulating Breg numbers in AS.

OBJECTIVES: Methotrexate (MTX) functions as an antiproliferative agent in cancer and an anti-inflammatory drug in rheumatoid arthritis (RA). Although macrophages critically contribute to RA pathology, their response to MTX remains unknown. As a means to identify MTX response markers, we have explored its transcriptional effect on macrophages polarised by GM-CSF (GM-MO) or M-CSF (M-MO), which resemble proinflammatory and anti-inflammatory macrophages found in RA and normal joints, respectively. METHODS: The transcriptomic profile of both human macrophage subtypes exposed to 50 nM of MTX under long-term and short-term schedules were determined using gene expression microarrays, and validated through quantitative real time PCR and ELISA. The molecular pathway involved in macrophage MTX-responsiveness was determined through pharmacological, siRNA-mediated knockdown approaches, metabolomics for polyglutamylated-MTX detection, western blot, and immunofluorescence on RA and normal joints. RESULTS: MTX exclusively modulated gene expression in proinflammatory GM-MO, where it influenced the expression of 757 genes and induced CCL20 and LIF at the mRNA and protein levels. Pharmacological and siRNA-mediated approaches indicated that macrophage subset-specific MTX responsiveness correlates with thymidylate synthase (TS) expression, as proinflammatory TS+ GM-MO are susceptible to MTX, whereas anti-inflammatory TSlow/- M-MO and monocytes are refractory to MTX. Furthermore, p53 activity was found to mediate the TS-dependent MTX-responsiveness of proinflammatory TS+ GM-MO. Importantly, TS and p53 were found to be expressed by CD163+/TNFalpha+ GM-CSF-polarised macrophages from RA joints but not from normal synovium. CONCLUSIONS: Macrophage response to MTX is polarisation-dependent and determined by the TS-p53 axis. CCL20 and LIF constitute novel macrophage markers for MTX responsiveness in vitro.

Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes (LNs) where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signalling mechanisms. We have analysed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking-down of AMPK with siRNA extends mDCs survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the LNs. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser485, which was mediated by Gi/Gbetagamma, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and Proximity Ligation Assays (PLA) indicate that AMPKalpha associate with ERK, but not with MEK. The results suggest that in addition to Akt-dependent signalling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signalling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response.

IntroductionCirculating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of follicular helper T cells (Tfh). There are three subpopulations of circulating Tfh (cTfh): CXCR5 inverted question mark+ inverted question markCXCR3 inverted question mark+ inverted question markCCR6- (Tfh-Th1), CXCR5 inverted question mark+ inverted question markCXCR3-CCR6- (Tfh-Th2) and CXCR5 inverted question mark+ inverted question markCXCR3-CCR6+ (Tfh-Th17). Our objective was to study the B cell helping capacity of cTfh subsets, and examine their frequency in Rheumatoid Arthritis (RA) patients, together with the frequency of circulating plasmablasts (CD19 inverted question mark+ inverted question markCD20-CD38high).MethodsPeripheral blood was drawn from RA patients with active disease (RA-a, DAS28 inverted question mark> inverted question mark2.6) (n inverted question mark= inverted question mark17), RA in remission (RA-r, DAS28 inverted question mark< inverted question mark2.6) (n inverted question mark= inverted question mark17) and healthy controls (HC) (n inverted question mark= inverted question mark34). cTfh and plasmablast frequencies were determined by flow cytometry. Cocultures of sorted CD4 inverted question mark+ inverted question markCXCR5+ T cell subpopulations were established with autologous CD19 inverted question mark+ inverted question markCD27- naive B cells of HC, and concentrations of IgG, A and M were measured in supernatants.ResultsIsolated Tfh-Th2 and Tfh-Th17 but not Tfh-Th1 cells, induced naive B cells to secrete IgG and IgA. The frequency of CXCR5+ cells gated for CD4+ T cells was not different among HC, RA-a and RA-r. In contrast, both RA-a and RA-r patients demonstrated an increased frequency of CD4 inverted question mark+ inverted question markCXCR5 inverted question mark+ inverted question markICOS+ T cells and augmented (%Tfh-Th2 inverted question mark+ inverted question mark%Tfh-Th17)/%Tfh-Th1 ratio as compared with HC. In addition, RA-a but not RA-r patients, showed an increased frequency of circulating plasmablasts.ConclusionBoth RA-a and RA-r patients demonstrate an increased frequency of cTfh and overrepresentation of cTfh subsets bearing a B cell helper phenotype, suggesting that altered germinal center dynamics play a role in RA pathogenesis. In contrast, only RA-a patients show an increased proportion of circulating plasmablasts.

Vitamin B9, commonly known as folate, is an essential cofactor for one-carbon metabolism that enters cells through three major specialized transporter molecules (RFC, FR, and PCFT), which differ in expression pattern, affinity for substrate, and ligand-binding pH dependency. We now report that the expression of the folate transporters differs between macrophage subtypes and explains the higher accumulation of 5-MTHF-the major folate form found in serum-in M2 macrophages in vitro and in vivo. M1 macrophages display a higher expression of RFC, whereas FRbeta and PCFT are preferentially expressed by anti-inflammatory and homeostatic M2 macrophages. These differences are also seen in macrophages from normal tissues involved in folate transit (placenta, liver, colon) and inflamed tissues (ulcerative colitis, RA), as M2-like macrophages from normal tissues express FRbeta and PCFT, whereas TNF-alpha-expressing M1 macrophages from inflamed tissues are RFC+. Besides, we provide evidences that activin A is a critical factor controlling the set of folate transporters in macrophages, as it down-regulates FRbeta, up-regulates RFC expression, and modulates 5-MTHF uptake. All of these experiments support the notion that folate handling is dependent on the stage of macrophage polarization.

Follicular helper T cells (Tfh), localized in lymphoid organs, promote B cell differentiation and function. Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of Tfh. Three subpopulations of circulating CD4+CXCR5+ cells have been described: CXCR3+CCR6- (Tfh-Th1), CXCR3-CCR6+ (Tfh-Th17), and CXCR3-CCR6- (Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 function as B cell helpers. Our objective was to study the frequencies of circulating Tfh (cTfh), cTfh subsets and plasmablasts (CD19+CD20-CD27+CD38high cells), and the function of cTfh cells, in patients with Ankylosing Spondylitis (AS). To this end, peripheral blood was drawn from healthy controls (HC) (n = 50), AS patients naive for TNF blockers (AS/nb) (n = 25) and AS patients treated with TNF blockers (AS/b) (n = 25). The frequencies of cTfh and plasmablasts were determined by flow cytometry. Cocultures of magnetically sorted CD4+CXCR5+ T cells with autologous CD19+CD27- naive B cells were established from 3 AS/nb patients and 3 HC, and concentrations of IgG, A and M were measured in supernatants. We obseved that AS/nb but not AS/b patients, demonstrated decreased frequencies of circulating CD4+CXCR5+ICOS+PD-1+ cells and plasmablasts, together with a decreased (Tfh-Th17+Tfh-Th2)/Tfh-Th1 ratio. The amounts of IgG and IgA produced in cocultures of CD4+CXCR5+ T cells with CD19+CD27- B cells of AS/nb patients were significantly lower than observed in cocultures established from HC. In summary, AS/nb but not AS/b patients, demonstrate a decreased frequency of cTfh and plasmablasts, and an underrepresentation of cTfh subsets bearing a B helper phenotype. In addition, peripheral blood CD4+CXCR5+ T cells of AS/nb patients showed a decreased capacity to help B cells ex vivo.