The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (e.g., TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (SIGLEC3, SIGLEC14, SIGLEC8), ribosomal proteins (RPL17, RPL34, RPS3A, MRPS33) and chemokines (CCL5, CCL24, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized non-coding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated TXNL1 gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including TXNL1, SIGLEC14, SIGLEC8, MRP4, and CA5A).

OBJECTIVE

Mesenchymal stem cells (MSCs) are potential effective therapy for tissue repair and bone regeneration. In present study, the effects of CXC chemokine ligand-13 (CXCL13) were evaluated on tendon-bone healing of rats.

METHODS

Tendon bone healing of the rat model was established and biomechanical testing was performed at 2, 4, 8 weeks after surgery. Murine mesenchymal cell line (C3HIOT1/2 cells) was cultured. The expression of miRNA-23a was detected by real-time PCR. The protein expression of ERK1/2, JNK and p38 was detected by western blotting. MiR-23a mimic and inhibitor were used to overexpress or silence the expression of miR-23a.

RESULTS

MSCs significantly elevated the levels of ultimate load to failure, stiffness and stress in specimens of rats, the effects of which were enhanced by CXCL13. The expression of miR-23a was down-regulated and the protein of ERK1/2 level was up-regulated by CXCL13 treatment in both in vivo and in vitro experiments. ERK1/2 expression was elevated by overexpression of miR-23a and reduced by miR-23a inhibitor.

CONCLUSIONS

These findings revealed that CXCL13 promoted the tendon-bone healing in rats with MSCs treatment, and implied that the activation of ERK1/2 via miR-23a was involved in the process of MSCs treated bone regeneration.

Secondary lymphoid organs (SLO) provide the structural framework for coconcentration of Ag and Ag-specific lymphocytes required for an efficient adaptive immune system. The spleen is the primordial SLO, and evolved concurrently with Ig/TCR:pMHC-based adaptive immunity. The earliest cellular/histological event in the ontogeny of the spleen's lymphoid architecture, the white pulp (WP), is the accumulation of B cells around splenic vasculature, an evolutionarily conserved feature since the spleen's emergence in early jawed vertebrates such as sharks. In mammals, B cells are indispensable for both formation and maintenance of SLO microarchitecture; their expression of lymphotoxin α1β2 (LTα1β2) is required for the LTα1β2:CXCL13 positive feedback loop without which SLO cannot properly form. Despite the spleen's central role in the evolution of adaptive immunity, neither the initiating event nor the B cell subset necessary for WP formation has been identified. We therefore sought to identify both in mouse. We detected CXCL13 protein in late embryonic splenic vasculature, and its expression was TNF-α and RAG-2 independent. A substantial influx of CXCR5(+) transitional B cells into the spleen occurred 18 h before birth. However, these late embryonic B cells were unresponsive to CXCL13 (although responsive to CXCL12) and phenotypically indistinguishable from blood-derived B cells. Only after birth did B cells acquire CXCL13 responsiveness, accumulate around splenic vasculature, and establish the uniquely splenic B cell compartment, enriched for CXCL13-responsive late transitional cells. Thus, CXCL13 is the initiating component of the CXCL13:LTα1β2 positive feedback loop required for WP ontogeny, and CXCL13-responsive late transitional B cells are the initiating subset.

Multiple myeloma (MM) is a hematologic cancer arising from plasma cells. Mesenchymal stem cells (MSCs) are a heterogeneous cell population in the bone marrow microenvironment. In this study, we evaluated the regulatory effects of MSCs on the invasion and drug resistance of MM cells U266 and LP-1. Bone marrow samples from MM patients and normal subjects were collected. MSCs were extracted from bone marrow and cultured, and their phenotypes were identified by flow cytometry. The level of CXCL13 in the supernatant of cultured MSCs was detected by ELISA. The protein expression of CXCR5 (a specific receptor of CXCL13) in U266 and LP-1 cells was detected by Western blot. The effects of MSCs on the invasion of U266 and LP-1 cells and the resistance to bortezomib were assessed by Transwell and CCK-8 assay, respectively. The mRNA and protein expressions of BTK, NF-κB, BCL-2, and MDR-1 were detected by RT-PCR and Western blot, respectively. CXCL13 was secreted by MSCs in the bone marrow microenvironment, and the level in MSCs from MM patients was significantly higher than that of healthy subjects. CXCR5 was expressed in both U266 and LP-1 cells. The resistance of MM cells to bortezomib was enhanced by MSCs through CXCL13 secretion. The invasion and proliferation of U266 and LP-1 cells were promoted, and the mRNA and protein expressions of BTK, NF-κB, BCL-2, and MDR-1 were upregulated by MSCs. The basic biological functions of MM cells U266 and LP-1 were affected by MSCs via the CXCL13-mediated signaling pathway. This study provides valuable experimental evidence for clinical MM therapy.

Background: Reversal of CD8 T-cell exhaustion was considered a major antitumor mechanism of anti-programmed cell death-1 (PD-1)/ anti-programmed death ligand-1 (PD-L1)-based immune checkpoint inhibitor (ICI) therapy.

Objectives: The aim of this study was to identify markers of T-cell exhaustion that is best associated with ICI treatment efficacy for advanced hepatocellular carcinoma (HCC).

Methods: Immune cell composition of archival tumor samples was analyzed by transcriptomic analysis and multiplex immunofluorescence staining.

Results: HCC patients with objective response after anti-PD-1/anti-PD-L1-based ICI therapy (n = 42) had higher expression of genes related to T-cell exhaustion. A 9-gene signature (LAG3, CD244, CCL5, CXCL9, CXCL13, MSR1, CSF3R, CYBB, and KLRK1) was defined, whose expression was higher in patients with response to ICI therapy, correlated with density of CD8⁺LAG3⁺ cells in tumor microenvironment, and independently predicted better progression-free and overall survival. This 9-gene signature had similar predictive values for patients who received single-agent or combination ICI therapy and was not associated with prognosis in HCC patients who received surgery, suggesting that it may outperform other T-cell signatures for predicting efficacy of ICI therapy for HCC. For HCC patients who underwent surgery for both the primary liver and metastatic lung tumors (n = 31), lung metastatic HCC was associated with a higher exhausted CD8 T-cell signature, consistent with prior observation that patients with lung metastatic HCC may have higher probability of response to ICI therapy.

Conclusions: CD8 T-cell exhaustion in tumor microenvironment may predict better efficacy of ICI therapy for HCC.

Keywords: Anti-PD-1; Anti-PD-L1; Immune checkpoint inhibitor; T-cell exhaustion; Tumor microenvironment.

In the era of primary vaccination against HPV and at the beginning of the low prevalence of cervical lesions, introduction of screening methods that can distinguish between low- and high-grade lesions is necessary in order to maintain the positive predictive value of screening. This case-control study included 562 women who attended cervical screening or were referred for colposcopy and 140 disease free controls, confirmed by histology and/or cytology. The cases were stratified by age. Using routine exfoliated liquid based cytological samples RT-PCR measurements of biomarker genes, high-risk HPV testing and liquid based cytology were performed and used to evaluate different testing protocols including sets of genes/tests with different test cut-offs for the diagnostic panels. Three new panels of cellular biomarkers for improved triage of hrHPV positive women (diagnostic panel) and for prognostic assessment of CIN lesions were proposed. The diagnostic panel (PIK3AP1, TP63 and DSG3) has the potential to distinguish cytologically normal hrHPV+ women from hrHPV+ women with CIN2+. The prognostic gene panels (KRT78, MUC5AC, BPIFB1 and CXCL13, TP63, DSG3) have the ability to differentiate hrHPV+ CIN1 and carcinoma cases. The diagnostic triage panel showed good likelihood ratios for all age groups. The panel showed age-unrelated performance and even better diagnostic value under age 30, a unique feature among the established cervical triage tests. The prognostic gene-panels demonstrated good discriminatory power and oncogenic, anti-oncogenic grouping of genes. The study highlights the potential for the gene expression panels to be used for diagnostic triage and lesion prognostics in cervical cancer screening.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

PD-1 inhibitors have been used to revive exhausted T cell responses in non-small cell lung cancer (NSCLC) and other malignancies. CXCR5⁺ T follicular helper (Tfh) cells are characterized by constitutive high PD-1 expression and have been associated with the formation of tertiary lymphoid structures and implicated in antitumor immunity. In this study, we investigated the effect of PD-1 and PD-1 inhibition on CXCR5⁺ CD4 T cells. Data showed that CXCR5⁺ CD4 T cells in both healthy subjects and NSCLC patients presented markedly higher PD-1 expression than CXCR5^- CD4 T cells. Both CXCR5^- and CXCR5⁺ CD4 T cells from NSCLC patients presented higher PD-1 expression than their counterparts in healthy subjects. PD-1⁺ CXCR5⁺ CD4 T cells were functional, could express IL-21, IL-10, and CXCL13 upon stimulation, demonstrated auxiliary effects toward CD8 T cell-mediated IFN-γ production and proliferation, and promoted IgM and IgG production. However, the potency of PD-1⁺ CXCR5⁺ CD4 T cells was lower than the potency of PD-1^- CXCR5⁺ CD4 T cells. PD-1 blocking could significantly enhance the effector functions of PD-1⁺ CXCR5⁺ CD4 T cells. Overall, this study demonstrated that PD-1⁺ CXCR5⁺ CD4 T cells could promote CD8 T cell and B cell inflammation and could be modulated by PD-1 inhibition.

BACKGROUND

There is a pressing need in rheumatoid arthritis (RA) to identify patients who will not respond to first-line disease-modifying anti-rheumatic drugs (DMARD). We explored whether differences in genomic architecture represented by a chromosome conformation signature (CCS) in blood taken from early RA patients before methotrexate (MTX) treatment could assist in identifying non-response to DMARD and, whether there is an association between such a signature and RA specific expression quantitative trait loci (eQTL).

METHODS

We looked for the presence of a CCS in blood from early RA patients commencing MTX using chromosome conformation capture by EpiSwitch™. Using blood samples from MTX responders, non-responders and healthy controls, a custom designed biomarker discovery array was refined to a 5-marker CCS that could discriminate between responders and non-responders to MTX. We cross-validated the predictive power of the CCS by generating 150 randomized groups of 59 early RA patients (30 responders and 29 non-responders) before MTX treatment. The CCS was validated using a blinded, independent cohort of 19 early RA patients (9 responders and 10 non-responders). Last, the loci of the CCS markers were mapped to RA-specific eQTL.

RESULTS

We identified a 5-marker CCS that could identify, at baseline, responders and non-responders to MTX. The CCS consisted of binary chromosome conformations in the genomic regions of IFNAR1, IL-21R, IL-23, CXCL13 and IL-17A. When tested on a cohort of 59 RA patients, the CCS provided a negative predictive value of 90.0% for MTX response. When tested on a blinded independent validation cohort of 19 early RA patients, the signature demonstrated a true negative response rate of 86 and a 90% sensitivity for detection of non-responders to MTX. Only conformations in responders mapped to RA-specific eQTL.

CONCLUSIONS

Here we demonstrate that detection of a CCS in blood in early RA is able to predict inadequate response to MTX with a high degree of accuracy. Our results provide a proof of principle that a priori stratification of response to MTX is possible, offering a mechanism to provide alternative treatments for non-responders to MTX earlier in the course of the disease.

Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c + dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 μM or 0.1 μM. Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of JAK2/1 inhibitors could be relevant to target lung macrophages in autoimmune and inflammatory pulmonary disorders that have no efficient disease modifying drugs to date.

Keywords: Inflammation; Interstitial Lung disease; Janus kinase inhibitors; Macrophage activation; Systemic sclerosis.

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterised by an increased risk for non-Hodgkin lymphoma (NHL) development. Ectopic germinal centre (GC) in the salivary gland is associated with increased NHL risk in pSS, and the chemokine CXCL13 is implicated in B-cell migration and GC formation. Serum CXCL13 concentrations were quantified by ELISA in 48 healthy individuals, 273 pSS patients without NHL (pSS-nonL), and 38 pSS patients with NHL (pSS-NHL+) from the United Kingdom Primary Sjögren's Syndrome Registry cohort. PSS-nonL patients were stratified into low risk (LR), moderate risk (MR) and high risk (HR) groups according to the lymphoma risk score proposed by Fragkioudaki et al. Differences in serum CXCL13 levels among groups were analysed using the Wilcoxon method. Also, changes in serum CXCL13 over a time period of at least 1 year and a median 4 years were assessed for 200 pSS-nonL and 8 pSS-NHL+ patients. In addition, associations of serum CXCL13 with B-cell and inflammatory markers were investigated by correlation analyses and logistic regression. Serum CXCL13 levels were higher in all pSS groups compared to controls (p < 0.0001), and in pSS-NHL+ compared to pSS-nonL patients (p = 0.0204). LR patients had lower CXCL13 levels than MR patients (p < 0.0001) and pSS-NHL+ patients (p = 0.0008). CXCL13 levels remained stable over the study period for all pSS groups. CXCL13 was associated (p < 0.0005) with Immunoglobulin G (IgG), B-cell activating factor, β2 microglobulin, combined free light chains, κ and λ light chains, anti-Ro/SSA, anti-La/SSB, and erythrocyte sedimentation rate. IgG and C3 controlled for age and gender were significantly associated with NHL risk in pSS. Serum CXCL13 levels were elevated in pSS-NHL+ and MR patients compared to LR patients and remained stable over time. Further study is required to investigate the role of CXCL13 in pSS-associated NHL risk.

While the contribution of autoreactive CD4⁺ T cells to the pathogenesis of Multiple Sclerosis (MS) is widely accepted, the advent of B cell-depleting monoclonal antibody (mAb) therapies has shed new light on the complex cellular mechanisms underlying MS pathogenesis. Evidence supports the involvement of B cells in both antibody-dependent and -independent capacities. T cell-dependent B cell responses originate and take shape in germinal centers (GCs), specialized microenvironments that regulate B cell activation and subsequent differentiation into antibody-secreting cells (ASCs) or memory B cells, a process for which CD4⁺ T cells, namely follicular T helper (T_FH) cells, are indispensable. ASCs carry out their effector function primarily via secreted Ig but also through the secretion of both pro- and anti-inflammatory cytokines. Memory B cells, in addition to being capable of rapidly differentiating into ASCs, can function as potent antigen-presenting cells (APCs) to cognate memory CD4⁺ T cells. Aberrant B cell responses are prevented, at least in part, by follicular regulatory T (T_FR) cells, which are key suppressors of GC-derived autoreactive B cell responses through the expression of inhibitory receptors and cytokines, such as CTLA4 and IL-10, respectively. Therefore, GCs represent a critical site of peripheral B cell tolerance, and their dysregulation has been implicated in the pathogenesis of several autoimmune diseases. In MS patients, the presence of GC-like leptomeningeal ectopic lymphoid follicles (eLFs) has prompted their investigation as potential sources of pathogenic B and T cell responses. This hypothesis is supported by elevated levels of CXCL13 and circulating T_FH cells in the cerebrospinal fluid (CSF) of MS patients, both of which are required to initiate and maintain GC reactions. Additionally, eLFs in post-mortem MS patient samples are notably devoid of T_FR cells. The ability of GCs to generate and perpetuate, but also regulate autoreactive B and T cell responses driving MS pathology makes them an attractive target for therapeutic intervention. In this review, we will summarize the evidence from both humans and animal models supporting B cells as drivers of MS, the role of GC-like eLFs in the pathogenesis of MS, and mechanisms controlling GC-derived autoreactive B cell responses in MS.

Keywords: B cell; ELF; GCR; TFH; Th17 (T helper 17 cell); ectopic lymphoid follicles; follicular T helper cells; germinal center.

Inducible bronchus-associated lymphoid tissue (iBALT) is a long lasting tertiary lymphoid tissue that can be induced following influenza A virus (IAV) infection. Previous studies have shown that iBALT structures containing germinal center (GC) B cells protect against repeated infection by contributing locally to the cellular and humoral immune response. If we are to exploit this in vaccination strategies, we need a better understanding on how iBALT structures are induced. One hypothesis is that the strength of the initial innate response dictates induction of iBALT. In the present study, we investigated the role of interleukin (IL)-1 and IL-1R signaling on iBALT formation. Mice lacking the IL-1R had a delayed viral clearance and, thus, a prolonged exposure to viral replication, leading to increased disease severity, compared to wild-type mice. Contradictorily, iBALT formation following clearance of the virus was heavily compromised in Il1r1 (-/-) mice. Quantification of gene induction after IAV infection demonstrated induction of IL-1α and to a much lesser extent of IL-1β. Administration of recombinant IL-1α to the lungs of wild-type mice, early but not late, after IAV infection led to more pronounced iBALT formation and an increased amount of GC B cells in the lungs. Bone marrow chimeric mice identified the stromal compartment as the crucial IL-1 responsive cell for iBALT induction. Mechanistically, Q-PCR analysis of lung homogenates revealed a strongly diminished production of CXCL13, a B cell-attracting chemokine, in Il1r (-/-) mice during the early innate phase of IAV infection. These experiments demonstrate that appropriate innate IL-1α-IL-1R signaling is necessary for IAV clearance and at the same time instructs the formation of organized tertiary lymphoid tissues through induction of CXCL13 early after infection. These findings are discussed in the light of recent insights on the pathogenesis of tertiary lymphoid organ formation in the lung in various diseases where the IL-1 axis is hyperactive, such as rheumatoid arthritis and COPD.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy diagnosed in children and is a malignant disorder that originates from one single hematopoietic precursor committed to B- or T-cell lineage. C-C chemokine receptor type 5 (CCR5) is a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. A highly potent competitive antagonist of CCR5, maraviroc, recently has been identified with suppression of cancer cells aggressive in a variety of cancers. However, the effects of maraviroc on ALL cells have not yet been defined. Here we report that CCR5 selective inhibitor significantly inhibited ALL cells SUP-B15 growth and induced SUP-B15 cells to undergo cell apoptosis. This cell apoptosis was associated with increased levels of cleavage of caspase-3 and caspase-9, and Poly (ADP-ribose) polymerase (PARP). Moreover, we demonstrated that maraviroc strongly inhibited SUP-B15 cells migration to C-X-C motif chemokine ligand 12 (CXCL12) and CXCL13, and adhesion to fibronectin and vascular cell adhesion molecule 1 (VCAM-1) in vitro. Importantly, CCR5-activated signaling proteins Janus Kinase 1 (JAK1), JAK2 and signal transducer and activator of transcription (STAT3) were inhibited by maraviroc. Finally, maraviroc suppressed the growth of SUP-B15 xenograft tumors in athymic mice. Collectively, this study demonstrated that CCR5 inhibition by maraviroc has the potential for the treatment of human ALL.

Recent progresses in the understanding of facioscapulohumeral muscular dystrophy (FSHD) genetics opened the way to the development of targeted therapies. However, knowledge about pathophysiology of muscle damage is still limited and there is increasing need to identify biomarkers of disease activity in the perspective of clinical trial readiness.We analyzed inflammatory mediators in the interstitial fluid of muscles with different MRI signal in FSHD patients, comparing muscles displaying early lesions on short-tau inversion recovery (STIR) sequences with normal ones. Patients with one T1-weighted normal and STIR hyperintense (STIR+) and contralateral T1-weighted and STIR normal (STIR-) lower limb muscle were asked to enter the study. Twelve consecutive patients, five controls, and one non-penetrant gene carrier underwent prolonged muscle microdialysis with high cut-off membranes. Microdialysates were analyzed using xMAP technology with a wide panel for cytokines, chemokines, and growth factors. A small number of inflammatory mediators were dysregulated in STIR+ versus STIR- and control muscles: CXCL13, upregulated in STIR+ muscles compared with controls (p < 0.01); CXCL5, downregulated in STIR+ compared with STIR- muscles (p < 0.05); and G-CSF, downregulated in STIR+ muscles compared with controls (p < 0.05). CXCL13 was also upregulated in the STIR+ muscles compared with the contralateral STIR- muscles of the same patient (p < 0.01).These results support the evidence of a selective inflammatory process taking place in STIR+ FSHD muscles. The application of microdialysis could provide insights on novel mechanisms involved in muscle damage in FSHD and in other myopathies. Further studies are needed to validate these investigated molecules as tissue and circulating biomarkers.

Persistent central nervous system (CNS) inflammation, as seen in chronic infections or inflammatory demyelinating diseases such as Multiple Sclerosis (MS), results in the accumulation of various B cell subsets in the CNS, including naïve, activated, memory B cells (Bmem), and antibody secreting cells (ASC). However, factors driving heterogeneous B cell subset accumulation and antibody (Ab) production in the CNS compartment, including the contribution of ectopic lymphoid follicles (ELF), during chronic CNS inflammation remain unclear and is a major gap in our understanding of neuroinflammation. We sought to address this gap using the Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model of progressive MS. In this model, injection of the virus into susceptible mouse strains results in a persistent infection associated with demyelination and progressive disability. During chronic infection, the predominant B cell phenotypes accumulating in the CNS were isotype-switched B cells, including Bmem and ASC with naïve/early activated and transitional B cells present at low frequencies. B cell accumulation in the CNS during chronic TMEV-IDD coincided with intrathecal Ab synthesis in the cerebrospinal fluid (CSF). Mature and isotype-switched B cells predominately localized to the meninges and perivascular space, with IgG isotype-switched B cells frequently accumulating in the parenchymal space. Both mature and isotype-switched B cells and T cells occupied meningeal and perivascular spaces, with minimal evidence for spatial organization typical of ELF mimicking secondary lymphoid organs (SLO). Moreover, immunohistological analysis of immune cell aggregates revealed a lack of SLO-like ELF features, such as cell proliferation, cell death, and germinal center B cell markers. Nonetheless, flow cytometric assessment of B cells within the CNS showed enhanced expression of activation markers, including moderate upregulation of GL7 and expression of the costimulatory molecule CD80. B cell-related chemokines and trophic factors, including APRIL, BAFF, CXCL9, CXCL10, CCL19, and CXCL13, were elevated in the CNS. These results indicate that localization of heterogeneous B cell populations, including activated and isotype-switched B cell phenotypes, to the CNS and intrathecal Ab (ItAb) synthesis can occur independently of SLO-like follicles during chronic inflammatory demyelinating disease.

Increasing evidence highlight the involvement of immune cells in brain activity and its dysfunction. The brain's immune compartment is a dynamic ensemble of cells that can fluctuate even in naïve animals. However, the dynamics and factors that can affect the composition of immune cells in the naïve brain are largely unknown. Here we examined whether acute sleep deprivation can affect the brain's immune compartment (parenchyma, meninges and choroid plexus). Using high-dimensional mass cytometry analysis we broadly characterized the effects of short-term sleep deprivation on the immune composition in the mouse brain. We found that after 6 hours of sleep deprivation there was a significant increase in the abundance of B cells in the brain compartment. This effect can be accounted for, at least in part, by the elevated expression of the migration-related receptor, CXCR5, on B cells and its ligand, cxcl13, in the meninges following sleep deprivation. Thus, our study reveals that short-term sleep deprivation affects the brain's immune compartment, offering a new insight into how sleep disorders can affect brain function and potentially contribute to neurodegeneration and neuroinflammation.

Marek's disease virus (MDV) is a cell associated alphaherpesvirus that causes fatal lymphoma in chickens. One factor that plays a crucial role in MDV pathogenesis is the viral CXC chemokine vIL-8 that was originally named after chicken interleukin 8 (cIL-8). However, a recent study demonstrated that vIL-8 recruits B cells and a subset of T cells but not neutrophils, suggesting that vIL-8 is not a cIL-8 orthologue. In this study, we set to identify the cellular orthologues and receptor of vIL-8 using in silico analyses, binding and chemotaxis assays. Sequence and phylogenetic analyses of all chicken CXC chemokines present in the recently published chicken genome revealed that vIL-8 shares the highest amino acid similarity with the CXCL13L1 variant. To evaluate if vIL-8 and CXCL13L1 are also functional orthologues, we assessed their binding properties and chemotaxis activity. We demonstrated that both vIL-8 and CXCL13 variants bind B cells and subsets of T cells, confirming that they target the same cell types. In addition, the chemokines not only bound the target cells but also induced chemotaxis. Finally, we identified CXCR5 as the receptor of vIL-8 and CXCL13 variants and confirmed that the receptor is expressed on MDV target cells. Taken together, our data demonstrate the conservation of the receptor-ligand interaction between CXCR5 and CXCL13 and shed light on the origin and function of the MDV-encoded vIL-8 chemokine, which plays a crucial role in the pathogenesis of this highly oncogenic virus.

Vaccination of neonates and young infants is hampered by the relative immaturity of their immune systems and the lack of safe and efficacious vaccine adjuvants. Immaturity of the follicular dendritic cells (FDCs), in particular, appears to play a critical role for the inability to stimulate immune responses. Using the CD21mT/mG mouse model we found that at 7 days of life, FDCs exhibited a mature phenotype only in the Peyer´s patches (PP), but our unique adjuvant, CTA1-DD, effectively matured FDCs also in peripheral lymph nodes following systemic, as well as mucosal immunizations. This was a direct effect of complement receptor 2-binding to the FDC and a CTA1-enzyme-dependent enhancing effect on gene transcription, among which CR2, IL-6, ICAM-1, IL-1β, and CXCL13 encoding genes were upregulated. This way we achieved FDC maturation, increased germinal center B-cell- and Tfh responses, and enhanced specific antibody levels close to adult magnitudes. Oral priming immunization of neonates against influenza infection with CTA1-3M2e-DD effectively promoted anti-M2e-immunity and significantly reduced morbidity against a live virus challenge infection. To the best of our knowledge, this is the first study to demonstrate direct effects of an adjuvant on FDC gene transcriptional functions and the subsequent enhancement of neonatal immune responses.

Spinal cord ischemia-reperfusion injury (SCII) remains an unresolved complication and its underlying mechanism has not been fully elucidated. In this study, we studied the role of chemokine (C-X-C motif) ligand 13 (CXCL13) in a rat model of SCII. We examined the time course and cellular distribution of CXCL13 protein in rats after SCII. The effects of siRNA targeting CXCL13 or C-X-C chemokine receptor type 5 (CXCR5) in SCII were also investigated. Neurological function, histological assessment, and disruption of the blood-spinal cord barrier (BSCB) were evaluated. The expression levels of CXCL13, CXCR5, phosphorylated extracellular signal-regulated kinase (p-ERK), caspase-3, interleukin 6 (IL-6), TNF-α, and IL-1β were determined. We found that SCII resulted in impaired hind limb function and increased the expression of CXCL13. In addition, CXCL13 expression demonstrated the most pronounced effect at 24 h after SCII. We reveal that CXCL13 protein was co-expressed with the mature neuron marker NeuN and the microglial marker IBA-1 in spinal cord tissues of model rats. SCII also increased the expression of CXCR5, p-ERK, caspase-3, IL-6, TNF-α, and IL-1β at 24 h after SCII. Pre-treatment with CXCL13 siRNA protected the rats against SCII and decreased the expression of signalling pathway proteins and proinflammatory cytokines mentioned above. CXCR5 siRNA also showed similar protective effects. These findings indicate that CXCL13 is involved in SCII. The CXCL13/CXCR5 axis promotes the development of SCII, possibly via ERK-mediated pathways. Targeting the mechanism of CXCL13 involved in the development of SCII might be a potential approach for the treatment of this condition.

For laboratory diagnostics of Lyme neuroborreliosis (LNB), the recomBead Borrelia antibody index (AI) assay has shown promising results in a mixed age population, but has not previously been evaluated with specific focus on paediatric patients. The aim of the study was to evaluate the recomBead Borrelia AI assay in cerebrospinal fluid (CSF) for the laboratory diagnosis of LNB in children. We also wanted to explore whether early markers, such as CXCL13 in CSF and/or total IgM index could be useful as complementary diagnostic tools. Children being evaluated for LNB in a Swedish Lyme endemic area were included in the study (n = 146). Serum and CSF were collected on admission. Patients with other specific diagnoses were controls (n = 15). The recomBead Borrelia AI assay and the recomBead CXCL13 assay (Mikrogen) were applied together with total IgM index. The overall sensitivity for recomBead Borrelia AI (IgM and IgG together) was 74% and the specificity was 97%. However, the highest sensitivity (91%) at an acceptable level of specificity (90%) was obtained by recomBead Borrelia AI together with CXCL13 and total IgM index, showing a positive predictive value of 84% and a negative predictive value of 95%. Thus, the recomBead Borrelia AI assay performs with moderate sensitivity and high specificity in paediatric LNB patients. The major advantage seems to be increased sensitivity in the possible LNB group compared to the IDEIA assay. The diagnostic sensitivity may be further increased by using a combination of early markers, such as CXCL13 in CSF and total IgM index.

Background: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) of HBV/HIV-1-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss.

Methods: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects who had been HBsAg-positive for ≥6 months before ART initiation and were followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV mono-infected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection.

Results: All observation period of ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed that IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF.

Conclusions: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients.

Keywords: Antiretroviral therapy; Chemokine; Chronic hepatitis; Functional cure; HBV/HIV-1.

The overall survival of metastatic colon adenocarcinoma (COAD) remains poor, so it is important to explore the mechanisms of metastasis and invasion. This study aimed to identify invasion-related genetic markers for prognosis prediction in patients with COAD. Three molecular subtypes (C1, C2, and C3) were obtained based on 97 metastasis-related genes in 365 COAD samples from The Cancer Genome Atlas (TCGA). A total of 983 differentially expressed genes (DEGs) were identified among the different subtypes by using the limma package. A 6-gene signature (ITLN1, HOXD9, TSPAN11, GPRC5B, TIMP1, and CXCL13) was constructed via Lasso-Cox analysis. The signature showed strong robustness and could be used in the training, testing, and external validation (GSE17537) cohorts with stable predictive efficiency. Compared with other published signatures, our model showed better performance in predicting outcomes. Pan-cancer expression analysis results showed that ITLN1, TSPAN11, CXCL13, and GPRC5B were downregulated and TIMP1 was upregulated in most tumor samples, including COAD, which was consistent with the results of the TCGA and GEO cohorts. Western blot analysis and immunohistochemistry were performed to validate protein expression. Tumor immune infiltration analysis results showed that TSPAN11, GPRC5B, TIMP1, and CXCL13 protein levels were significantly positively correlated with CD4+ T cells, macrophages, neutrophils, and dendritic cells. Further, the TIMP1 and CXCL13 proteins were significantly related to the tumor immune infiltration of CD8+ T cells. We recommend using our signature as a molecular prognostic classifier to assess the prognostic risk of patients with COAD.

Keywords: Colon carcinoma; Gene signature; Invasion-related genes; Molecular subtyping; Prognosis.