Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes.

Background: Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype. Methods: HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice. Results: Cobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN. Conclusion: Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.

Comprehensive BCMA Expression Profiling in Adult Normal Human Brain Suggests a Low Risk of On-target Neurotoxicity in BCMA-targeting Multiple Myeloma Therapy.

B-cell maturation antigen (BCMA) is a target for the treatment of multiple myeloma with cytolytic therapies, such as chimeric antigen receptor T-cells or T-cell redirecting antibodies. To better understand the potential for "on-target/off-tumor" toxicity caused by BCMA-targeting cytolytic therapies in the brain, we investigated normal brain BCMA expression. An immunohistochemistry (IHC) assay using the E6D7B commercial monoclonal antibody was applied to 107 formalin-fixed, paraffin-embedded brain samples (cerebrum, basal ganglia, cerebellum, brainstem; 63 unique donors). Although immunoreactivity was observed in a small number of neurons in brain regions including the striatum, thalamus, midbrain, and medulla, this immunoreactivity was considered nonspecific and not reflective of BCMA expression because it was distinct from the membranous and Golgi-like pattern seen in positive control samples, was not replicated when a different IHC antibody (D6 clone) was used, and was not corroborated by in situ hybridization data. Analysis of RNA-sequencing data from 478 donors in the GTEx and Allen BrainSpan databases demonstrated low levels of BCMA RNA expression in the striatum of young donors with levels becoming negligible beyond 30 years of age. We concluded that BCMA protein is not present in normal adult human brain, and therefore on-target toxicity in the brain is unlikely.

Inadequate use of antibiotics in the covid-19 era: effectiveness of antibiotic therapy.

BACKGROUND: Since December 2019, the COVID-19 pandemic has changed the concept of medicine. This work aims to analyze the use of antibiotics in patients admitted to the hospital due to SARS-CoV-2 infection. METHODS: This work analyzes the use and effectiveness of antibiotics in hospitalized patients with COVID-19 based on data from the SEMI-COVID-19 registry, an initiative to generate knowledge about this disease using data from electronic medical records. Our primary endpoint was all-cause in-hospital mortality according to antibiotic use. The secondary endpoint was the effect of macrolides on mortality. RESULTS: Of 13,932 patients, antibiotics were used in 12,238. The overall death rate was 20.7% and higher among those taking antibiotics (87.8%). Higher mortality was observed with use of all antibiotics (OR 1.40, 95% CI 1.21-1.62; p < .001) except macrolides, which had a higher survival rate (OR 0.70, 95% CI 0.64-0.76; p < .001). The decision to start antibiotics was influenced by presence of increased inflammatory markers and any kind of infiltrate on an x-ray. Patients receiving antibiotics required respiratory support and were transferred to intensive care units more often. CONCLUSIONS: Bacterial co-infection was uncommon among COVID-19 patients, yet use of antibiotics was high. There is insufficient evidence to support widespread use of empiric antibiotics in these patients. Most may not require empiric treatment and if they do, there is promising evidence regarding azithromycin as a potential COVID-19 treatment.

Headache during SARS-CoV-2 infection as an early symptom associated with a more benign course of disease: a case-control study.

BACKGROUND AND PURPOSE: Headache is an important manifestation during SARS-CoV-2 infection. In this study, the aim was to identify factors associated with headache in COVID-19 and headache characteristics. METHODS: This case-control study includes COVID-19 hospitalized patients with pneumonia during March 2020. Controls comprise COVID-19 patients without headache and the cases are COVID-19 patients with headache. Demographic, clinical and laboratory data were obtained from the medical records. Headache characteristics were evaluated by semi-structured telephonic interview after discharge. RESULTS: Of a total of 379 COVID-19 patients, 48 (13%) developed headache. Amongst these, 30 (62%) were men and the median age was 57.9 (47-73) years. Headache was associated with younger age, fewer comorbidities and reduced mortality, as well as with low levels of C-reactive protein, mild acute respiratory distress syndrome and oropharyngeal symptoms. A logistic multiple regression model revealed that headache was directly associated with D-dimer and creatinine levels, the use of high flow nasal cannula and arthromyalgia, whilst urea levels, beta-lactamic treatment and hypertension were negatively associated with headache. COVID-19-associated headache characteristics were available for 23/48 (48%) patients. Headache was the onset symptom in 8/20 (40%) patients, of mild or moderate intensity in 17/20 (85%) patients, with oppressive characteristics in 17/18 (94%) and of holocranial 8/19 (42%) or temporal 7/19 (37%) localization. CONCLUSIONS: Our results show that headache is associated with a more benign SARS-CoV-2 infection. COVID-19-associated headache appears as an early symptom and as a novel headache with characteristics of headache attributed to systemic viral infection. Further research addressing the underlying mechanisms to confirm these findings is warranted.

Design of an anti-inflammatory diet (ITIS diet) for patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects synovial joints, leading to inflammation, joint destruction, loss of function, and disability. Although recent pharmaceutical advances have improved treatment of RA, patients with RA often inquire about dietary interventions to improve RA symptoms, as they perceive rapid changes in their symptoms after consumption of certain foods. There is evidence that some ingredients have pro- or anti-inflammatory effects. In addition, recent literature has shown a link between diet and microbiome changes. Both diet and the gut microbiome are linked to circulating metabolites that may modulate inflammation. However, evidence of the effects of an anti-inflammatory and probiotic-rich diet in patients with RA is scarce. There is also a need for biological data to support its anti-inflammatory effects. METHODS: The main goal of this study is to delineate the design process for a diet tailored to our RA population. To achieve this goal, we collected information on diet, supplements, cooking methods, and intake of different ingredients for each patient. Different groups were interviewed, and their feedback was assessed to design a diet that incorporates suggested anti-inflammatory ingredients in a manner that was easy for patients to adopt based on their lifestyles and backgrounds. RESULTS: We designed a diet that includes a high intake of potential anti-inflammatory ingredients. Feedback from highly motivated patients was critical in constructing an anti-inflammatory diet (ITIS diet) with elevated adherence. CONCLUSION: In order to tailor our diet, we surveyed our patients on several different parameters. We obtained important feedback on how feasible our ITIS diet is for RA patients. Using this feedback, we made minor improvements and finalized the design of the ITIS diet. This diet is being used in an on-going pilot study to determine their anti-inflammatory effect in pain and joint swelling in RA patients. TRIAL REGISTRATION: Not applicable.

Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis.

OBJECTIVES: Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage. METHODS: HK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells. RESULTS: HK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage. CONCLUSION: HK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Fibroblast-like synoviocyte metabolism in the pathogenesis of rheumatoid arthritis.

An increasing number of studies show how changes in intracellular metabolic pathways alter tumor and immune cell function. However, little information about metabolic changes in other cell types, including synovial fibroblasts, is available. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are the most common cell type at the pannus-cartilage junction and contribute to joint destruction through their production of cytokines, chemokines, and matrix-degrading molecules and by migrating and invading joint cartilage. In this review, we show that these cells differ from healthy synovial fibroblasts, not only in their marker expression, proto-oncogene expression, or their epigenetic changes, but also in their intracellular metabolism. These metabolic changes must occur due to the stressful microenvironment of inflamed tissues, where concentrations of crucial nutrients such as glucose, glutamine, and oxygen are spatially and temporally heterogeneous. In addition, these metabolic changes will increase metabolite exchange between fibroblast and other synovial cells, which can potentially be activated. Glucose and phospholipid metabolism as well as bioactive lipids, including sphingosine-1-phosphate and lysophosphatidic acid, among others, are involved in FLS activation. These metabolic changes likely contribute to FLS involvement in aspects of immune response initiation or abnormal immune responses and strongly contribute to joint destruction.

Pharmacogenomic study in patients with multiple sclerosis: Responders and nonresponders to IFN-ß.

OBJECTIVES: We aimed to investigate the association between polymorphisms located in type I interferon (IFN)-induced genes, genes belonging to the toll-like receptor (TLR) pathway, and genes encoding neurotransmitter receptors and the response to IFN-ß treatment in patients with multiple sclerosis (MS). METHODS: In a first or screening phase of the study, 384 polymorphisms were genotyped in 830 patients with MS classified into IFN-ß responders (n = 416) and nonresponders (n = 414) according to clinical criteria. In a second or validation phase, the most significant polymorphisms associated with IFN-ß response were genotyped in an independent validation cohort of 555 patients with MS (281 IFN-ß responders and 274 nonresponders). RESULTS: Seven single nucleotide polymorphisms (SNPs) were selected from the screening phase for further validation: rs832032 (GABRR3; p = 0.0006), rs6597 (STUB1; p = 0.019), rs3747517 (IFIH1; p = 0.010), rs2277302 (PELI3; p = 0.017), rs10958713 (IKBKB; p = 0.003), rs2834202 (IFNAR1; p = 0.030), and rs4422395 (CXCL1; p = 0.017). None of these SNPs were significantly associated with IFN-ß response when genotyped in an independent cohort of patients. Combined analysis of these SNPs in all patients with MS (N = 1,385) revealed 2 polymorphisms associated with IFN-ß response: rs2277302 (PELI3; p = 0.008) and rs832032 (GABRR3; p = 0.006). CONCLUSIONS: These findings do not support an association between polymorphisms located in genes related to the type I IFN or TLR pathways or genes encoding neurotransmitter receptors and the clinical response to IFN-ß. Nevertheless, additional genetic and functional studies of PELI3 and GABRR3 are warranted.

NLRP3 inflammasome is associated with the response to IFN-ß in patients with multiple sclerosis.

Evidence exists for a potential modulation of inflammasome activity by interferon beta. Here, we investigated the roles of inflammasomes [absent in melanoma 2 (AIM2); NLR family, CARD domain containing 4 (NLRC4); NLR family, pyrin domain containing 1 and 3 (NLRP1 and NLRP3)] and related cytokines (IL1B, IL10, IL18) in the response to interferon beta in patients with relapsing-remitting multiple sclerosis. Ninety-seven patients treated with interferon beta were classified into responders and non-responders according to clinical criteria after 24 months and clinical-radiological criteria after 12 months of treatment. Messenger RNA expression levels of inflammasomes and cytokines were determined by real-time polymerase chain reaction in peripheral blood mononuclear cells collected before treatment with interferon beta. In a subgroup of patients, NLRP3 and IL1B expression was also determined after 3 months (n = 32) and 12 months (n = 20) of interferon beta treatment. A polymorphism located in the NLRP3 gene, rs35829419, was genotyped in 789 multiple sclerosis patients treated with interferon beta. Baseline mRNA expression levels for NLRP3 and IL1B were increased in peripheral blood mononuclear cells from non-responders compared to responders classified according to clinical criteria after 24 months (P = 0.02 and P = 0.001, respectively). No significant differences were observed for other inflammasomes and related cytokines. Differences in NLRP3 and IL1B expression remained significant following a clinical-radiological classification after 12 months (P = 0.007 and P = 0.02, respectively). After treatment with interferon beta, NLRP3 and IL1B expression was increased in responders but unchanged in non-responders. A trend for association was observed between rs35829419 and interferon beta response (pM-H = 0.08). These results point to a role of the NLRP3 inflammasome and its related cytokine IL1B in the response to interferon beta in patients with relapsing-remitting multiple sclerosis.

Activation-induced cell death in T lymphocytes from multiple sclerosis patients.

Apoptosis is a major mechanism regulating immune tolerance by the elimination of autoreactive T lymphocytes. A failure of activation induced cell-death (AICD) has been described in T lymphocytes from patients with multiple sclerosis (MS). The aims of this study were to evaluate AICD in T lymphocytes from patients with MS and healthy controls, and to explore the molecular mechanisms underlying the deregulation observed in apoptosis induction. PHA-induced AICD was reduced in T lymphocytes from patients with relapsing-remitting MS compared with controls. This finding was associated with a diminished expression of Fas and a failure in caspase 3 activation.

Baseline gene expression signatures in monocytes from multiple sclerosis patients treated with interferon-beta.

BACKGROUND: A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. METHODS: Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. RESULTS AND DISCUSSION: Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb.

Treatment with MOG-DNA vaccines induces CD4+CD25+FoxP3+ regulatory T cells and up-regulates genes with neuroprotective functions in experimental autoimmune encephalomyelitis.

BACKGROUND: DNA vaccines represent promising therapeutic strategies in autoimmune disorders such as multiple sclerosis (MS). However, the precise mechanisms by which DNA vaccines induce immune regulation remain largely unknown. Here, we aimed to expand previous knowledge existing on the mechanisms of action of DNA vaccines in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), by treating EAE mice with a DNA vaccine encoding the myelin oligodendrocyte glycoprotein (MOG), and exploring the therapeutic effects on the disease-induced inflammatory and neurodegenerative changes. METHODS: EAE was induced in C57BL6/J mice by immunization with MOG35₋55 peptide. Mice were intramuscularly treated with a MOG-DNA vaccine or vehicle in prophylactic and therapeutic approaches. Histological studies were performed in central nervous system (CNS) tissue. Cytokine production and regulatory T cell (Treg) quantification were achieved by flow cytometry. Gene expression patterns were determined using microarrays, and the main findings were validated by real-time PCR. RESULTS: MOG-DNA treatment reduced the clinical and histopathological signs of EAE when administered in both prophylactic and therapeutic settings. Suppression of clinical EAE was associated with dampening of antigen (Ag)-specific proinflammatory Th1 and Th17 immune responses and, interestingly, expansion of Treg in the periphery and upregulation in the CNS of genes encoding neurotrophic factors and proteins involved in remyelination. CONCLUSIONS: These results suggest for the first time that the beneficial effects of DNA vaccines in EAE are not limited to anti-inflammatory mechanisms, and DNA vaccines may also exert positive effects through hitherto unknown neuroprotective mechanisms.

Implication of the Toll-like receptor 4 pathway in the response to interferon-ß in multiple sclerosis.

OBJECTIVE: Interferon-beta (IFNß) has demonstrated beneficial effects reducing disease activity in multiple sclerosis (MS) patients, but a relatively large proportion of patients do not respond to treatment. Here we aimed to investigate the roles of the Toll-like receptor 4 (TLR4) and the type I IFN pathways in the response to IFNß in MS patients. METHODS: The expression levels of several components of the TLR4 and the type I IFN pathways were determined by flow cytometry and real-time polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMCs) from a cohort of 85 MS patients treated for at least 2 years with IFNß and classified into responders, intermediate responders, and nonresponders based on their clinical response to treatment. Thirty-two healthy controls were also included in the study for comparison purposes. RESULTS: Compared to responders and controls, PBMCs from nonresponders and intermediate responders were characterized by increased baseline expression levels of endogenous IFNß and elevated IFN receptor 1 (IFNAR1) expression in monocytes. Furthermore, the capacity of IFNß to induce its own expression was deficient in cells from nonresponders compared with responders. Baseline expression of the interleukin-1 receptor-associated kinase 3 (IRAK3), a negative regulator of TLR4 signaling primarily expressed in monocytes, was found to be significantly decreased in IFNß responders compared with nonresponders. INTERPRETATION: These findings provide evidence of the involvement of the TLR4 and type I IFN signaling pathways in the response to IFNß.

Search for specific biomarkers of IFNß bioactivity in patients with multiple sclerosis.

Myxovirus A (MxA), a protein encoded by the MX1 gene with antiviral activity, has proven to be a sensitive measure of IFNß bioactivity in multiple sclerosis (MS). However, the use of MxA as a biomarker of IFNß bioactivity has been criticized for the lack of evidence of its role on disease pathogenesis and the clinical response to IFNß. Here, we aimed to identify specific biomarkers of IFNß bioactivity in order to compare their gene expression induction by type I IFNs with the MxA, and to investigate their potential role in MS pathogenesis. Gene expression microarrays were performed in PBMC from MS patients who developed neutralizing antibodies (NAB) to IFNß at 12 and/or 24 months of treatment and patients who remained NAB negative. Nine genes followed patterns in gene expression over time similar to the MX1, which was considered the gold standard gene, and were selected for further experiments: IFI6, IFI27, IFI44L, IFIT1, HERC5, LY6E, RSAD2, SIGLEC1, and USP18. In vitro experiments in PBMC from healthy controls revealed specific induction of selected biomarkers by IFNß but not IFNγ, and several markers, in particular USP18 and HERC5, were shown to be significantly induced at lower IFNß concentrations and more selective than the MX1 as biomarkers of IFNß bioactivity. In addition, USP18 expression was deficient in MS patients compared with healthy controls (p = 0.0004). We propose specific biomarkers that may be considered in addition to the MxA to evaluate IFNß bioactivity, and to further explore their implication in MS pathogenesis.

Gender-associated differences of perforin polymorphisms in the susceptibility to multiple sclerosis.

The granule-dependent exocytosis pathway is an important mechanism to induce apoptosis by CD8(+) T cells and NK cells and involves lytic molecules such as perforin. In the current study, we investigated the perforin 1 gene (PRF1) as a candidate for multiple sclerosis (MS) susceptibility in the Spanish population. We genotyped three PRF1 single nucleotide polymorphisms (rs885822, rs10999426, and rs3758562) in 420 patients with MS and 512 controls. Associations of PRF1 polymorphisms with the disease were restricted to male patients with MS, and the finding was consistently observed at the allele, genotype, and haplotype levels. Gender-associated differences were validated in an additional replication cohort comprised of 292 MS cases and 300 controls. In addition, we identified minor risk haplotypes strongly associated with male patients having primary progressive MS (PPMS). Further characterization of male patients with PPMS carrying the risk haplotypes by means of gene expression microarrays revealed overrepresentation of the cell killing gene ontology category among downregulated genes in these patients compared with male patients with PPMS carrying protective haplotypes. Moreover, PRF1 mRNA expression levels were significantly lower in patients with risk haplotypes, and changes in perforin protein expression by CD8(+) T cells mirrored those observed in gene expression. These findings suggest a gender dimorphism in the PRF1 association with MS and point to the presence of a generalized defect in the expression of genes that code for proteins involved in cell killing in a subgroup of male patients with PPMS.