The 2019 coronavirus disease (COVID-19) infection had newly emerged with predominant respiratory complications. Other extrapulmonary features had been recently described. Here, we describe a COVID-19 patient presenting with multiorgan involvement mimicking systemic lupus erythematosus. He was successfully treated with glucocorticoids and tocilizumab. Coronavirus disease (COVID-19) uses angiotensin converting enzyme 2 as its functional receptor to infect endothelial target cells in lung, kidney, liver, heart, and the gastrointestinal tract. Therefore, in addition to respiratory symptoms that usually dominate the clinical presentation, there is recent increasing data showing that this novel coronavirus has potential extrapulmonary complications. Here, we report the case of the COVID-19 infection with multiorgan dysfunction, simulating systemic lupus erythematosus (SLE) presentation.A 62-year-old man with no medical history was referred by his primary care physician to our Department of Internal Medicine on April 4, 2020, for shortness of breath associated with a kidney injury. He suffered from fever, cough, and myalgia 17 days before. On examination, the temperature was 39°C, the blood pressure 105/85 mm Hg, and the heart rate 97 beats per minute. His oxygen saturation was 94% on room air. He had bibasilar rales. A neuropsychiatric examination showed acute confusion, disorientation in time and space, and behavioral disorders. He had dysexecutive syndrome consisting of slowed thinking, information processing impairment, memory loss, inattention, and poorly and slowed organized movements in response to command.3, lymphopenia at 800/mm3, 0 eosinophils per mm3, mildly increased liver enzymes, elevated C-reactive protein (CRP) concentration and procalcitonin at 208 mg/l and 2.41 ng/ml, respectively. Protein electrophoresis showed hypoalbuminemia at 19.7 g/l and elevated α 2 globulin at 11.8 g/l. He had a blood urea nitrogen at 7 µmol/L, blood creatinine at 204 µmol/L with an estimated glomerular filtration rate (EGFR) at 38 mL/min/1.73 m2. His baseline creatinine level was unknown. A urinalysis showed the protein-to-creatinine ratio at 233 mg/mmol with no evidence of hematuria, sodium 9 mmol/L, and potassium 36 mmol/L. Blood and urine cultures were negative. The laboratory results are shown in Laboratory tests showed the white blood cell count at 10 900/mmInvestigations for mycoplasma pneumoniae, human immunodeficiency virus, influenza A and B, hepatitis A, B, and C, and antineutrophil cytoplasmic antibodies were negative. Serum Complement 3 and 4 levels were normal. Antinuclear antibodies (ANA) were positive at 1/160. Anti-double stranded (ds) DNA and anti-extractable nuclear antigen antibodies were negative. A cerebrospinal fluid (CSF) examination and brain magnetic resonance imaging were normal. A lung computed tomography (CT) imaging revealed extensive lesions with ground-glass opacities, consolidations, and a crazy paving pattern with the extent of the lesions at 25% A. A CT sThe patient was started on empiric antibiotics (Rovamycine and ceftriaxone) and prophylactic anticoagulation. Three days after, the oxygen flow rate was progressively increased to 6 L/min. He had tachycardia at 120/min. His renal function worsened with his respiratory distress. Blood creatinine continued to rise to 325 µmol/L. His urine output remained between 1500 and 1800 ml/day. He was given curative anticoagulation due to pulmonary embolism suspicion. Angio CT of the thorax was not performed due to his renal function deterioration. He received progressive intravenous hydration with little improvement of his creatinine level that subsequently stabilized at 260 µmol/L.2, urine sodium: 40 mmol/l, urine potassium: 29 mmol/l) with a protein-to-creatinine ratio at 200 mg/mmol, hematuria at 200/mm3, and leukocyturia at 100/mm3. Urine culture remained negative. An angio CT of the thorax, abdomen, and pelvis showed significant worsening of pulmonary lesions extent between 50 and 75%  and anti-dsDNA antibody test are more specific than ANA for lupus nephritis, which is usually associated with complement consumption. Therefore, the age and male gender of our patient, elevated CRP and procalcitonin, low ANA titer, negative anti-dsDNA and anti Sm, and normal serum complement level called into doubt the diagnosis of SLE. The diagnosis of COVID-19 with multiorgan damages was more probable.Lupus-like syndrome is usually induced by drugs, more rarely by infections activating autoimmune response by transient positive autoantibodies, which can be associated with lupus-like symptoms such as malar rash, arthralgia, nephritis, and acute hepatitis.2 In Cheng et al.’s large prospective cohort study including 701 patients with COVID-19,3 the prevalence of proteinuria, hematuria, EGFR under 60 ml/min/1.73m2, and AKI occurring during the study period was 43.9%, 26.7%, 13.1%, and 5.1%, respectively. A Chinese report on a renal histopathological analysis of 26 autopsies of patients with COVID-19 revealed acute proximal tubular injury, vacuolar degeneration, erythrocyte aggregation in peritubular and glomerular capillaries, glomerular fibrin thrombi, ischemic changes, and severe endothelial injury.4 COVID-19 particles were identified in the cytoplasm of renal proximal tubular epithelium, as well as in the podocytes with positive COVID-19 nucleoprotein in tubules by an indirect fluorescence method. Interestingly, rare cases of glomerulonephritis in patients with COVID-19 have been described.5Recently, some publications have illustrated the renal invasive potential of COVID-19. Notably, acute kidney injury (AKI) is noted in 9.2% of population in South Korea.6 Our patient had no cardiovascular factors risk. His volume contraction due to decreased oral input and insensible fluid loss associated with fever contributed to AKI, which explained little improvement of his renal function after fluid resuscitation. His urine dipstick test was compatible with glomerular syndrome and probable tubular disorders. Unfortunately, he did not have a renal biopsy because of safety concerns.In addition to direct virus mediated injury, kidney involvement with a COVID-19 infection could be due to systemic hypoxia, hypercoagulability, microangiopathy, rhabdomyolysis, sepsis, cytokine storm, and angiotensin II pathway activation.7 encephalitis, and Guillain Barré syndrome. In Helms et al.’s report,8 including 58 patients hospitalized in the intensive care unit (ICU), confusion was noted in 26 of 40 patients and dysexecutive syndrome in 15 of 45 (33%), such as in the case of our patient.There is also an increasing evidence of central neurological complications of COVID-19, including headaches, acute stroke,7 Renal involvement is considered a new predictor of patient deterioration. It correlates with a poor prognosis and is associated with a higher risk of mortality.3Kidney and neurological complications were described in critically ill patients with COVID-19.10 The most used medications in COVID-19 patients with renal complications are diuretics, glucocorticoids, antivirus, and renal replacement therapy.3 To our knowledge, preliminary data on TCZ use in kidney complications are very limited. Interestingly, our patient was successfully treated with glucocorticoids and TCZ, preventing admission in ICU with significant improvement of kidney, respiratory, neurological, and liver damages.In recent reports, TCZ has been used in patients with COVID-19 and severe pulmonary involvement showing encouraging results with reduced ventilatory support requirements, especially in those with cytokine storm and higher IL-6 levels.COVID-19 can induce kidney, neurological, liver, and vascular damage with possible positive ANA simulating multisystem inflammatory disease. Renal dysfunction is rare during this infection and usually associated with a fatal clinical course. Fortunately, our patient was discharged from the hospital with favorable outcome after glucocorticoids and TCZ treatment. Therefore, regulatory monitoring of inflammatory parameters, creatinine, and urine sample analysis allows an early disease worsening prediction leading to appropriate therapeutic options onset in critically ill patients.


Kidney function was assessed as estimated glomerular filtration rate (eGFR). Calcium/creatinine ratio (Ca/Cr) was used as index of urinary calcium in timed overnight urine under fed condition (Gubbio study), morning urine after overnight fast (Gubbio study), and first-void morning urine (Moli-sani study). Moli-sani study included also data for glomerular filtered calcium load, tubular calcium handling, and serum phosphorus, parathyroid hormone, 1,25-dihydroxyvitamin D, calcium, and 25-hydroxyvitamin D. eGFR positively and independently related to Ca/Cr (p < 0.001). In multivariate analyses, eGFR lower by 10 mL/min × 1.73 m2 related to overnight urine Ca/Cr lower by 14.0 mg/g in men and 17.8 mg/g in women, to morning urine Ca/Cr lower by 9.3 mg/g in men and 11.2 mg/g in women, and to first-void urine Ca/Cr lower by 7.7 mg/g in men and 9.6 mg/g in women (p < 0.001). eGFR independently related to glomerular filtered calcium load (p < 0.001) and did not relate to tubular calcium handling (p ≥ 0.35). In reduced eGFR only (<90 mL/min × 1.73 m2), low urine Ca/Cr independently related to low serum 1,25-dihydroxyvitamin D (p = 0.002) and did not relate to hyperphosphatemia, high serum parathyroid hormone, or hypocalcemia (p ≥ 0.14). Population-based data indicated consistent associations of lower kidney function with lower urine calcium due to reduction in glomerular filtered calcium. In reduced kidney function, relative hypocalciuria associated with higher prevalence of low serum 1,25-dihydroxyvitamin D.This observational, cross-sectional, epidemiological analysis investigated relationships of kidney function to urine calcium and other variables. The analyses targeted two population-based samples of adults (Gubbio study and Moli-sani study:  The CKDium (Ca) . The curium (Ca) . The guiurine Ca ,4. In 20 formula . In 2019rmula 2 ,6, hence, hence2Drmula [2 . Data onrmula [2 . The preThe Gubbio study is a population-based cohort study ongoing since 1982 in the city of Gubbio, Italy ; early m2D [2D were performed by a fully automated chemiluminescent assay  [The Moli-sani study is a prospective cohort study ongoing since 2005 that enrolled 24,325 individuals from 2005 to 2010, men and women, aged ≥35 years, randomly recruited from the general population of a region of central-southern Italy . The stu2D . Automat2D ,17. Seru2D . The mea, Italy) ,18. As p, Italy) ,19, the , Italy) . The dat, Italy) . This va, Italy) . Of the Kidney function was assessed as eGFR calculated by the Chronic Kidney Disease—Epidemiology Collaboration equation with the use of serum creatinine, ethnicity, sex, and age . Both inp ≥ 4.5 mg/100 mL, high serum PTH as serum PTH ≥ 66 pg/mL, and low serum 1,25(OH) 2D as 1,25(OH) 2D < 18 pg/mL [Kidney function was assessed as eGFR calculated by the Chronic Kidney Disease—Epidemiology Collaboration equation including serum creatinine, ethnicity, sex, age, and cystatin C also to reduce the confounding of creatinine generation ,32. Urin18 pg/mL .2. eGFR < 90 mL/min × 1.73 m2 was defined as reduced kidney function [eGFR was the main independent variable in both datasets and was divided in the following seven strata: ≥90, 89–75, 74–60, 59–45, 44–30, 29–15, and <15 mL/min × 1.73 mfunction . First, function . As anci2. In both datasets, there were positive trends along eGFR strata for female sex prevalence, age, and body mass index , with a difference in morning fasting urine Ca/creatinine ratio of 9.3 mg/g in men and of 11.2 mg/g in women , and with a difference in first-void urine Ca/creatinine ratio of 7.7 mg/g in men and of 9.6 mg/g in women . Results were similar in analyses controlled also for additional variables  that was the strongest correlate of 25(OH)D .For serum Ca, lower eGFR associated with weak trends to lower total Ca and higher ultra-filterable Ca . The conp < 0.001) but similar prevalence of high serum PTH , of low serum 1,25(OH)2 , and of hypocalcemia . In uni-variate analyses, reduced kidney function associated not significantly with higher prevalence of high serum PTH, low serum 1,25(OH)2D and hypocalcemia among men, and with higher prevalence of hyperphosphatemia, high serum PTH, and low serum 1,25(OH)2D among women . Findings were not significant for the relation of urine Ca/creatinine ratio to hyperphosphatemia, high serum PTH, and hypocalcemia calcemia .2D.This observational, population-based, cross-sectional analysis reported two main findings. First, there is a continuous, graded, and independent association of lower kidney function with lower urine Ca and, as expected, with lower glomerular filtered load of Ca, in the absence of alterations in the tubular Ca handling. Second, in individuals with reduced kidney function, the relative hypocalciuria independently associates with increased prevalence of low serum 1,25(OH)2D in combination with higher fibroblast growth factor 23 [2D secondary to kidney hypofunction.The interpretation of observational, cross-sectional data should be cautious. Study results indicated that a reduction in the glomerular filtered Ca load was the sole mechanism responsible for the relative hypocalciuria associated with reduced kidney function, at least over the eGFR range explorable in the two population samples in analysis. This interpretation was coherently supported by different observations: the consistence of results in urine samples under fed condition and after prolonged fast, the independence of findings of dietary markers, Ca supplementation, and duration of overnight fast. The lack of altered tubular Ca handling in reduced kidney function appeared coherent with the concurrence of conditions capable either to up-regulate and to down-regulate tubular Ca reabsorption as expected for higher serum PTH and of lower serum 1,25(OH)actor 23 . In a fi2D [Regarding metabolic indices of CKD-MBD, study results were coherent with the concept that a chronic reduction in kidney function induces an increase in serum levels of phosphorus and PTH and a decrease in serum 1,25(OH)2D .Study limitations were the use of the Ca/creatinine ratio as index of urinary Ca, the lack of 24-h urine collection, the lack of direct measurements of serum ultra-filterable Ca, the lack of standardized Ca intake, the low number of individuals with severe eGFR reduction, and the lack of data for different ethnic groups. The bias due to the use of the Ca/creatinine ratio was reasonably minor considering that the results in the Gubbio cohort for overnight urine were substantially identical with the use of the urine Ca/creatinine ratio and of the timed urine Ca excretion rate. The use of overnight urine instead of 24-h urine could have caused a bias due a circadian variation in creatinine excretion although overnight urine Ca strongly correlated with same day 24-h urine Ca also with urine Ca factored by urine creatinine .The study had some merits. The population-based design reduced the bias due to low sample size. The use of datasets from temporally and geographically separated epidemiological projects reduced the possibility of local or temporal confounders and proved the reproducibility of the findings. The collection of urine under different conditions proved that findings were independent of the time and the type of urine sample. The use of Ca/creatinine ratio might have ruled out the bias due to inaccuracies in timing and completeness of urine collections . Lastly,2D and less consistent association with serum Ca [Regarding the association of reduced kidney function with relative hypocalciuria, study results were in accordance with those derived from clinical settings where data were collected under different experimental conditions ,4. For bserum Ca ,36,38. Fserum Ca  and wereserum Ca ,41.2D deficiency associated with reduced kidney function. Data indicated that the assessment of urine Ca in patients with reduced kidney function could be of use as a supportive evidence of low 1,25(OH)2D and/or for monitoring treatment with calcium or vitamin D.In conclusion, this observational, cross-sectional, epidemiological study showed a continuous, graded, and independent relationship between kidney function and urine Ca. The relationship was reproducible in two different population samples, independently of the timing and of the type of the urine collection. The relative hypocalciuria associated with reduced kidney function appeared due to a reduction in the normal glomerular filtered Ca load. It could represent a beneficial adaptation to prevent or to reduce Ca deficiency secondary to the 1,25(OH)2D given that, in individuals with reduced kidney function, the prevalence of this metabolic abnormality was progressively higher with reducing the level of urine Ca independently of sex, age, weight, and eGFR. In addition, low urine Ca in reduced kidney function highlights the need of being cautious in the use of Ca supplementation to avoid excessively positive Ca balance in patients with reduced kidney function [The main practical implication of the present study is that the assessment of urine Ca could be added to the list of non-invasive analytes used in the diagnosis and monitoring of CKD-MBD . In partfunction .


IL6, IL8, LIF, MCP1, and TGFB1. On the other hand, infection by defective EV strains obtained from diabetic subjects suppressed IFN pathways and the transcription of most cytokines, while enhancing the expression of IL8, IL18, IL32, and MCP1. IL18 and IL32 are known for their pathogenic role in autoimmune diabetes. Thus, the cytokine profile of AV3 cells infected by diabetes-derived EV strains closely matches that observed in patients at the early stages of T1D. The concordance of our results with clinically verified information reinforces the hypothesis that the immune changes observed in type 1 diabetic patients are due to a hardly noticeable virus infection.Enterovirus (EV) infection of insulin-producing pancreatic beta cells is associated with type 1 diabetes (T1D), but little is known about the mechanisms that lead the virus to cause a persistent infection and, possibly, to induce beta cell autoimmunity. A cell line susceptible to most enterovirus types was infected with EV isolates from cases of T1D and, for comparison, with a replication-competent strain of coxsackievirus B3. The transcription of immune-related genes and secretion of cytokines was evaluated in infected vs. uninfected cells. Acutely infected cells showed the preserved transcription of type I interferon (IFN) pathways and the enhanced transcription/secretion of  Type 1 diabetes (T1D) is characterized by an irreversible impairment of glucose homeostasis due to the loss of insulin-producing pancreatic beta cells. Though T1D is considered a disease of the young, currently it is diagnosed in all decades of life. The disease originates from a complex interaction of genetic, immunological, and environmental factors such as virus infection of beta cells in the islets of Langerhans. Numerous studies have associated enterovirus (EV) infection with T1D, especially regarding the direct effect of the virus on pancreatic islets and its possible role in triggering islet-specific autoimmunity.EV antigens have been detected in pancreatic islets of post-mortem T1D cases . At the The results of collaborative studies within the network of Pancreatic Organ Donors with Diabetes  have reinforced this notion showing that EV infection subsists in the lymphoid and pancreatic tissues of T1D patients independently of time from diagnosis .Since the disease is, in part, immune-mediated, it is central to identify how the infecting viruses may trigger autoimmunity. A variety of mechanisms may operate . In the Within the islets, macrophages and dendritic cells are engaged in an inflammatory process in which many components of the immune system play a role (insulitis). Within endocrine epithelial cells, HLA-I molecules present antigens to immune cells and play a role in antiviral defense . SimilarIP-10, ISG15, OAS1, OAS2, Mx2), and different cytokines (interleukin-6 (IL6), IL8, monocyte chemoattractant protein-1 (MCP1)) [Regarding EV infection, innate immunity factors and cytokines are expressed in human cells challenged by coxsackievirus B3 (an enterovirus of the B species). Infection may be followed by the upregulation of viral RNA sensors, IFN-stimulated genes ( (MCP1)) . Studies in humans have shown that T1D is associated with a chronic type of EV infection ,15. ChroTo investigate the consequences of infection by EV strains obtained from T1D cases, we analyzed the transcriptome of a reporter epithelial cell line infected with virus isolates from T1D cases. Transcripts of immune-related genes were compared in infected vs. non-infected cells. Results indicate that infection by diabetes-derived EV strains influences multiple pathways. Under persistent infection, the presentation of autoantigens may initiate an autoimmune response ,18.Instrumentation, consumables, cell lines, culture media, chemicals, molecular biology reagents, antibodies, commercial kits, software and applications are listed in For virus isolation, spleen samples from the nPOD biobank  were shi2 using Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium supplemented with L-glutamine, heat-inactivated 10% fetal bovine serum (FBS), and penicillin/streptomycin. Cell cultures were checked monthly for mycoplasma contamination (MycoAlert Plus mycoplasma kit). The Nancy strain of coxsackievirus B3 (CBV3) was originally obtained from American Type Culture Collection , propagated in AV3 cells, titrated, and stored at −70 °C. CBV3 was used as a reference strain for transcriptome analysis since this virus is pancreotropic and diabetogenic in mice [AV3 cells (containing HeLa marker chromosomes and derived via HeLa contamination) and the RD, 1.1B4, VC3, and HEK-293 cell lines were obtained from The European Collection of Authenticated Cell Cultures (ECACC) and cultured at 37 °C in air with 5% CO in mice ,21,22 an in mice  as well  in mice . Regardi in mice ,26. For For indirect immunofluorescence, cell monolayers in Millicell EZ 4-well glass slides  were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde. The expression of EV receptors was investigated by indirect immunofluorescence in uninfected AV3 cells using the antibodies listed in A published procedure for isolating and detecting EV strains causing chronic infection was followed . BrieflyFor gene expression analysis, total RNA was purified from two T25 flasks containing monolayers of uninfected and infected AV3 cells using the RNeasy Mini Kit . RNA quality was assessed by spectrophotometry . A gene expression assay was carried out as previously reported  using thg for 5 min), and stored at −70 °C. Supernatants were then examined for levels of 22 human cytokines using Luminex xMAP Technology assays capable of measuring GM-GSF, IFN-alpha, IFN-gamma, IL1-beta, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL17, IL18, MIP1-alpha, MIP1-beta, CCL2/MCP1, CCL5/RANTES, MIG, TNF-alpha, TNF-beta .Supernatants of AV3 cell monolayers in T-25 flasks were collected from uninfected and infected cultures, incubated for 72 h, centrifuged to eliminate cell debris . The normalization of target counts was performed by background thresholding as previously reported . SimilarSpleen tissue from the nPOD biobank was used as a source for isolating T1D-associated defective EV strains. As shown in The expression of EV receptor molecules in AV3 cells was analyzed by indirect immunofluorescence. AV3 cells express high levels of poliovirus receptor (PVR), coxsackie-adenovirus receptor (CAR), decay-accelerating factor (DAF), scavenger receptor class B member 2 (SCARB2), and integrin alpha-V/beta-3 (ITGB3) as well as low levels of P-selectin glycoprotein ligand-1 (PSGL-1) and integrin alpha-V/beta-6 . In contrast, the major receptor for A-B rhinoviruses A and B, intercellular adhesion molecule-1 (ICAM-1), and the echovirus-1 receptor VLA-2 are not expressed . Figure AV3 cells were infected either with replication-competent CBV3 (CBV3) or with replication-defective EV strains from the spleen of donors with diabetes (Spl-EV). The latter strains were replicating very poorly and at minimal titers. However, though no manifest cytopathic effects were produced, the viral genome and viral antigens were consistently detected in cell cultures. There was no detection of 5’ terminal deletions in diabetes-derived EV strains but, as reported , scatterTwo experimental models were analyzed: (i) acute infection by CBV3, i.e., cells infected with a replication-competent CBV3 strain vs. uninfected control cells (CBV3 vs. CBV3-Ctrl); (ii) slow infection by EV isolates from T1D cases (Spl-EV) vs. mock-infected control cells (Spl-EV vs. Spl-Ctrl). Three independent experiments were performed. In each experiment, the CBV3 model was run in triplicate whereas the spleen-EV model was run in quadruplicate. Transcriptome results of the four experimental groups are available in After normalization and thresholding, 243 genes were selected for further analyses. Principal component analysis (PCA) showed that the first two principal components accounted for over 80% of variation. These were used for plotting. IFNAR1, IFNAR2) were upregulated together with cell adhesion molecules  and integrins , regulators of apoptosis and cell survival , the ETS1 transcription factor that controls the expression of cytokines and chemokines, and components of the proteasome (PSMB5) and of autophagic vesicles (ATG5). Notably, the expression of SLC2A1  was also enhanced in infected cells, possibly to sustain the energy levels adequate for virus production. Among cytokines, IL6, IL6 signal transducer (IL6ST), leukemia inhibitory factor (LIF), and transforming growth factor beta 1 (TGFB1) were upregulated. Transcription of the following genes was reduced: BCL3 , CEBPB (regulator of genes involved in immune and inflammatory responses), KLRAP1 (activation of NK cells), PTPN6 (regulator of innate immunity), and IL1A .In cells acutely infected with CBV3, 58 genes were differentially expressed a, with aJAK1, STAT1 and IFNAR1 were upregulated together with the adhesion molecule CD164, integrins ITGA6 and ITGAE, CASP3, ETS1, PSMB5, and SMAD5 . Selected cytokines were also upregulated: the macrophage chemotactic factor MCP1, IL18, and IL32. Downregulation was seen for TICAM1 (adapter for toll-like receptors to mediate NFKB and IRF activation and to induce apoptosis), IKBKG , TMEM173/STING1 (receptor that detects cytosolic nucleic acids and activates IFN-I), SOCS3 , and TAPBP (transport of antigenic peptides for peptide loading on MHC class I molecules).In the slow infection (Spl-EV vs. Spl-Ctrl), 94 genes were differentially expressed b with a IFIH1 was retained in both acute and slow infection. In acute infection, TRAF3 (a regulator of NFKB and MAP kinases) and IKB kinase alpha (IKBKA) were enhanced. Downregulation was seen only for IKB kinase beta (IKBKB). In the paracrine IFN pathway, the IFN receptors IFNAR1 and IFNAR2 were conserved or potentiated in acute infection. JAK1 and STAT1 transcription was also potentiated, showing that the infected cells remain capable of binding type I IFN. In contrast, cells infected with defective EV strains showed reduced transcription of the receptor molecule IFNAR1 and also of JAK2, TYK2, and STAT2 [Type I IFN pathways were compared in the acute and slow infection models . The cytnd STAT2 . Thus, cThe gage package was used to analyze the expression patterns of overlapping genes and their associated pathways. In CBV3-infected cells, the JAK-STAT signaling pathway was activated. In cells infected by Spl-EV, four different pathways were activated: B-cell receptor signaling, T-cell receptor signaling, neurotrophin signaling, and regulation of actin cytoskeleton. The activated pathways are summarized in PLAU) was seen, concomitantly with the activation of complement factor I (CFI), CF1, CFB, CFD, CF2, CF3, DAF, MCP, and vitronectin (VTN). Gene expression changes were interpreted as follows: since in spleen-derived preparations the virus was present in minimal amounts, the preparations had to be employed at 1:10 dilution (v/v) thus exposing cells to minute debris. Nonetheless, the DEGs induced by these preparations (In cells exposed to the spleen-derived preparations (either containing or not containing the virus) the coagulation and complement cascades were activated. Upregulation of urokinase-type plasminogen activator  and TGFB1 were upregulated. In the slow infection model, the downregulation of cytokines was predominant . The transcription of the negative regulators of cytokines SOCS3 and CISH (cytokine-inducible SH2-containing protein) was also reduced, possibly with a pro-viral role. Conversely, the transcription of IL18, IL32 and MCP1 was enhanced. The latter cytokines have a proinflammatory role, stimulate NK activity, increase the production of IFN-gamma, and are chemotactic for monocytes and basophils.In the acute infection model, Data were confirmed, in part, by measuring the release of cytokines in infected vs. uninfected cells. Infection significantly influenced the release of only 4 out of the 22 measured cytokines: IL6, IL8, IL18, and MCP-1 . CompareA wide array of EV receptors are expressed in the AV3 cell line . This liThe study aimed to check whether persistent EV infection could change the expression of immune genes in ways capable of promoting inflammation and the response to self-antigens. Comparing acute CBV3 infection to the slow infection caused by diabetes-related EV strains revealed interesting differences.Upon the recognition of viral pathogens by RNA sensors that include MDA-5 and TLR3, type I IFNs are produced. The binding of enteroviral RNA by MDA5 results in its association with mitochondrial antiviral signaling adaptor protein (MAVS) that binds to the TNF receptor-associated factor 3 (TRAF3). TRAF3 recruits and activates TANK-binding kinase 1 (TBK1) and inducible IKB kinase (IKK), leading to the phosphorylation of IFN regulatory factor 3 (IRF3) resulting in its translocation to the nucleus and transcription of IFN genes.JAK1 and STAT1 was enhanced both in the acute and the slow infection models. Inversely, in cells infected by defective EV strains nearly all components of the IFN induction pathway were downregulated, including IRF3. In the paracrine IFN signaling pathway, the transcription of IFNAR1, JAK2, TYK2 and STAT2 was reduced. Thus, infection by diabetes-related EV strains harmed both the IFN-inducing and the IFN-responsive pathways, furthering viral persistence [In acutely infected cells, the IFN induction pathway was preserved. Components of the IFN signaling pathway (i.e. the ability to respond to extracellular IFN) were not only preserved at the transcription level, but potentiated. Notably, the transcription of sistence ,32.In agreement with our observations, previous studies showed that picornaviruses are equipped to interfere with the IFN-mediated antiviral state not only at the transcriptional level, but also at the protein level. The Theiler virus  has been shown to interfere with transcription at two steps: (a) phosphorylation of IRF3 by TBK1 and IKKE, a process involving a complex with TRAF3; (b) nuclear entry downstream of IRF3 and binding to the IFN-beta gene promoter upstream of IRF3 . In addiPrevious studies of acute enteroviral infection showed that: (a) cultured neural cells infected with CBV3 had enhanced secretion of IL6, IL8, and MCP1 and the upregulation of SOCS3, a suppressor of cytokine signaling ; (b) humIL6, IL8, LIF, MCP1, and TGFB1. On the other hand, infection by diabetes-related EV strains reduced the expression of most cytokines including IFNs and IL6, but enhanced the expression of IL8, IL18, IL32, and MCP1. Thus, the expression of IL18 and IL32 may distinguish slow infection by diabetes-related EV strains from the acute model of infection.Our results on acute CBV3 infection are in line with the observations summarized above. In fact, acutely infected cells showed enhanced transcription and/or secretion of IL32 transcripts before the appearance of T1D-associated autoantibodies [Interestingly, pathogenetic studies of T1D—a disease in which persistent enteroviral infections are suspected—have provided results compatible with those observed for slow infection due to diabetes-related EV strains. Diabetes studies have indicated that: (a) IL6 is normally expressed in islet beta and alpha cells, but its production is reduced in the pancreas of diabetics ; (b) eletibodies . Finallytibodies . MCP-1 otibodies .In closing, the cytokine profile of AV3 cells infected by diabetes-related EV strains closely matches what is observed in patients at the early stages of T1D. Hence, our results reinforce the rationale that a long-standing viral infection of beta cells (perhaps in a slowly replicating form) may be one of the mechanisms leading to progressive beta cell loss in type 1 diabetes.Our study has two major limitations: (a) a transformed epithelial cell line not adequately mimicking pancreatic beta cells was used as reporter, and (b) only a few diabetes-related EV strains have been examined so far. Large studies might clarify whether, upon persistent infection, the variation of immune gene expression is linked either to differences among the diabetes-derived isolates or to other factors. The nPOD biobank is an abundant source of multiple tissues for studying diabetes cases in comparison to non-diabetic controls. With these limitations in mind, transcriptome analysis appears a valuable tool for analyzing the consequences of viral infection in diseases of unknown etiology.


It has been observed that obese lupus patients tend to have a have a worsening of their chronic inflammatory status, probably because of increased oxidative stress, increased C-reactive protein expression and elevated protein oxidation and lipid hydroperoxidation.3 The metabolic syndrome (MetS) is recognized as a chronic proinflammatory and prothrombotic state that aggravates insulin resistance, oxidative injury, and cardiovascular risk. It includes several risk factors such as central obesity, insulin resistance, dyslipidaemia, increased blood pressure, and endothelial dysfunction.4 The tissue possibly responsible for these events is the white adipose tissue that becomes dysfunctional and produces excessive amounts of pro-inflammatory soluble mediators including interleukin 6 (IL-6), and adipokines such as leptin, adiponectin, and resistin.5Mechanisms of systemic lupus erythematosus (SLE) exacerbation are still unclear. Several lines of evidence pointed out that metabolic syndrome is a frequent manifestation in SLE patients, with approximately 35% being overweight and another 27% obese.6 Among TLRs, some have been specifically evaluated in the context of obesity, such as TLR2, TLR6, and TLR7 that showed a lower expression after a high fat diet in the adipose tissue.7Toll-like receptors (TLRs) have been recently emerged as main actors in this pantomime playing a role linking inflammation, obesity and insulin resistance, both in humans and in murine models.8TLR7 gained attention also because it was shown to promote the effector functions of purified CD8+ T cells in vitro and the upregulation of glucose uptake and glycolysis.9Wong et al. in an extensive review on the argument, underlined how metabolic syndrome, TLR expression and inflammation are linked in a way that MetS triggers TLR activation via several mechanisms including AGEs, FFAs, HMGB1, HSPs, and endotoxins, and TLR activation in turn leads to inflammation via MyD88 and TRIF dependent pathways.10 This mouse spontaneously develops lupus-like disease due to increased TLR7 signalling by dendritic cells (DCs). Those mice fed with a HFD had an aggravated lupus condition characterized by increased overall immune activation, anti-DNA autoantibody production, and IgG/IgM glomerular deposition that were coupled with increased kidney histopathology.Recently, Hanna Kazazian N. and colleagues described in their paper a possible implication of TLR7 signalling in high-fat diet (HFD)-induced metabolic syndrome and exacerbation of lupus autoimmunity in a mouse model of lupus, the TLR8-deficient (TLR8ko) mice.Also, liver inflammation was worsened by HFD which may explain at least partly the relatively frequent sub-clinical liver inflammation that can be found in patients with SLE. T cells seem to be the major driver of such phenomena; in particular, HFD led to a reduction of splenic CD4+ and CD8+ T cells both in WT and TLR8ko mice, compared to standard-diet (SD)-fed mice, while the authors did not observe any difference between SD- and HFD-fed TLR7/8ko mice, concluding that both in the spleen and liver, HFD leads to an increase of effector memory and decrease of naïve CD4+ and CD8+ T cells in a TLR7-dependent manner. In conclusion, it seemed that upon HFD, TLR7/8ko mice did not develop SLE and both TLR7ko, and TLR7/8ko mice were fully protected from metabolic abnormalities, including body weight gain, insulin resistance, and liver inflammation. All these data together may suggest that TLR7 might be a novel approach as a tailored therapy in SLE and metabolic diseases.11It is noteworthy in this context that a very recent paper linked the interferon pathway with increased adipose tissue and metabolic inflammation. These authors found that adipose IRF5 gene and protein expression was up-regulated in obese compared with lean individuals and IRF5 gene expression was associated positively with adipose inflammatory signatures including local expression of TNF-α, IL-6, CXCL8, CCL-2/5, IL-1β, IL-18, CXCL-9/10, CCL7, CCR-1/2/5, IRF3, MyD88, IRAK-1, and inflammatory macrophage markers and, importantly, also with TLR-2/7/8/9.12 and the improvement goes together with a lower atherogenic risk due to reduced expression of IL-12 and IFN-γ, reduced NF-κB activation, and the induction of T regulatory cells.13 It is likely that a tailored diet, possibly based on the genetic study of the affected individual, may improve through TLR7, the outcome of lupus disease.Thus, the question is whether SLE can be improved through diet, including calories restriction or microbiota modification. Mouse models of SLE have proved so,


Molecular chaperones are a set of conserved proteins that have evolved to assist the folding of many newly synthesized proteins by preventing their misfolding under conditions such as elevated temperatures, hypoxia, acidosis and nutrient deprivation. Molecular chaperones belong to the heat shock protein (HSP) family. They have been identified as important participants in immune functions including antigen presentation, immunostimulation and immunomodulation, and play crucial roles in metabolic rewiring and epigenetic circuits. Growing evidence has accumulated to indicate that metabolic pathways and their metabolites influence the function of immune cells and can alter transcriptional activity through epigenetic modification of (de)methylation and (de)acetylation. However, whether molecular chaperones can regulate metabolic programs to influence immune activity is still largely unclear. In this review, we discuss the available data on the biological function of molecular chaperones to immune responses during inflammation, with a specific focus on the interplay between molecular chaperones and metabolic pathways that drive immune cell fate and function.  Molecular chaperones play a pivotal role in the maintenance of cellular proteostasis by preventing the misfolding and aggregation of nascent polypeptides by ensuring proper protein folding ,2. AlthoImmune cells are highly heterogeneous in population and dynamic in functional activities. They adopt distinct metabolic states to support both the energetic and biosynthetic demands of a range of processes . In geneMolecular chaperones have been widely investigated in regard to cancer metabolism and the inflammatory microenvironments, where they act as signals to regulate immunity and inflammation. Several studies have demonstrated that molecular chaperones could affect epigenetic variation to influence numerous transcriptional programs contributing to the phenotypic plasticity of cells ,13. The Drosophila larvae upon exposure to increased temperature [In the early 1960s, Ferruccio Ritossa observed the formation of new puffs in the chromosomes of perature . In 1970perature . Later, perature . A subseperature ; howeverperature . Now, moperature . Often, perature . Indeed,perature . HSR occperature . There aperature .In general, stress proteins are believed to act as intracellular molecular chaperones expressed in the cytosol and organelles such as mitochondria, endoplasmic reticulum (ER) and nucleus. Under certain circumstances, they are released to extracellular space and can serve as danger signals, directly stimulating innate immune cells. Indeed, HSP60 and HSP70 have been revealed to bind to toll-like receptor (TLR)2 and TLR4 to activate the downstream signaling pathway for eliciting innate immune responses ,27. In aS-adenosylmethionine (SAM) and nicotinamide adenine dinucleotide (NAD+). For instance, the modification of DNA and histone methylation requires SAM metabolite as the methyl donor used by the DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs) in cells, respectively. In contrast, the regulation of demethylation is tailored by ten-eleven translocation (TET) enzymes and histone demethylases (HDMs) using α-KG as a cofactor [Metabolic pathways are intimately linked to immune function ,32,33. Scofactor ,39. NumeThe members of the HSP90 family are highly conserved proteins that are localized in different cellular compartments. Two HSP90 isoforms, HSP90α (inducible form) and HSP90β (constitutively expressed form) are localized in the cytosol. Other family members including glucose-regulated protein 94 (GRP94) are localized in the ER, while tumor necrosis factor receptor-associated protein 1 (TRAP1) is mainly localized in the mitochondria ,41. HSP9HSP90 chaperones have been shown to induce innate and adaptive immunity by stimulating APCs and facilitating the activation and maturation of lymphocytes . SecreteIn past years, numerous reports have suggested that HSP90 chaperones can control metabolic rewiring in cancer cells for tumorigenesis ,60,61. H+ T cell activation [Moreover, HSP90 has been demonstrated to interact with and stabilize hypoxia-inducible factor 1-alpha (HIF-1α) ,69 Figu. HIF-1α tivation ,77. HoweIt has been suggested that HSP90 activity may influence phenotypic plasticity through epigenetic modification . NotablyTRAP1, a mitochondrial heat shock protein in the HSP90 family, controls a myriad of physiological roles, including cell proliferation, differentiation and survival ,85,86. IChaperone proteins of the HSP70 family are found in all organisms from archaebacteria and plants to humans. The human HSP70 family includes at least eight members. HSP70 protein 5  and HSP70-9 (known as mtHSP70 or GRP75) can be found in the ER lumen and the mitochondrial matrix, respectively, and the remaining six HSP70 proteins in the cytosol and nucleus ,91. HSP7+ T cell infiltration and anti-tumor immunity with macrophages and to suppress tumor growth, highlighting the potential for HSP70-targeted therapy as an efficacious anti-cancer strategy [Recently, it has been found that HSP70 exerts distinct expression patterns in tissue specific macrophages including microglia, monocytes, peritoneal macrophages and adipose tissue macrophages (ATMs) upon metabolic stress . Specifistrategy .Further, HSP70 presents immunomodulatory roles in T cells in which HSP70 treatment can elevate the suppressive capacity of Tregs by increasing the production of IL-10 and TGF-β and decreasing the proinflammatory cytokines TNF-α and IFN-γ . In cont2+ signaling between these two organelles and control bioenergetics, cell survival and cell death decisions [HSP70 has been shown to control metabolic processes such as mitochondrial respiration, leading to a switch in energy metabolism with a preference towards increased glycolytic metabolism . HSP70 hecisions , suggestecisions . These f+CD25+FoxP3+ regulatory T cells via innate TLR2 signaling [+ T cells from patients with remitting juvenile idiopathic arthritis express higher Th2 marker CD30 responding to HSP60 and can produce regulatory cytokines including IL-10 [HSP60 family members are present in the cytosol, mitochondria, and cell surface . HSP60 iignaling . Moreoveignaling ,141. CD4ng IL-10 , indicatFinally, HSP60 is a quality control protein in the mitochondria and is responsible for maintaining mitochondrial fitness . The dysSalmonella to ensure survival within macrophages and invasion of epithelial cells, leading to systemic infection [Streptococcus pneumoniae contributes to bacterial survival and elicits innate immune responses by inducing IL-6 production via the PI3K/c-Jun N-terminal kinase (JNK) signaling in macrophages [Streptococcus pneumoniae infections by promoting the production of IL-10, IFN-γ and IL-17A, as well as increased IgG titers and lymphocyte proliferation in mice in vivo [+ and CD8+ T cells and stimulates the secretion of IL-10 by PBMCs [HSP40  is involved in protein refolding, translocation and degradation, mainly by working as a cofactor of HSP70 ,147. HSPnfection . HSP40 srophages . Based o in vivo ,151. Alsby PBMCs . In addiby PBMCs ,154  are characterized as small molecular weight proteins between 12 and 43 kDa, containing a core α-crystallin domain (ACD) flanked by variable N-terminal and C-terminal domains and lack an ATPase domain . In gene-/- mice exhibit worse EAE symptoms, with higher Th1 and Th17 cytokine secretion from T cells and macrophages and severe CNS inflammation compared with the wildtype controls. However, the administration of recombinant HSPB5 protein ameliorates EAE, suggesting an immunosuppressive and therapeutic role for HSPB5 during autoimmune demyelination [2+ to prevent the generation of ROS and oxidative stress, leading to cytoprotective effects in neurodegenerative diseases [In mammalian cells, ten sHSPs (HSPB1 to HSPB10) have been identified. These are HSPB1 (HSP27), HSPB4 , HSPB5 , HSPB6 (HSP20) and HSPB8 (HSP22) or Class I sHSPs, and HSPB2, HSPB3, αA-crystallin, HSPB7, HSPB9 and HSPB10 or Class II sHSPs. Class I sHSPs are widely distributed in various tissues and play roles in cell survival during stress, while Class II sHSPs have tissue-specific expression and are involved in tissue development and differentiation . Also, slination . Similarlination . The immlination , and implination . It has diseases . Additiodiseases .2+ uptake, thus decreasing necrosis and apoptosis induced by ischemia/reperfusion (I/R) [+ T cells [Another class I sHSP HSPB1 can induce the production of IL-10 via activation of p38 signaling in monocytes , and exoon (I/R) . Notablyon (I/R) . Peroxison (I/R) ,176,177.on (I/R) ,179. Per T cells ,181. Als T cells ,183. Thi+ T cell-mediated immunity [Besides the intracellular functions performed by HSPs, several HSP can be secreted into extracellular space in various pathological conditions. HSP90, HSP70, HSP60, HSP40DnaJ, HSP47 and sHSP as mentioned above have been found outside cells in cancer, neurodegenerative and inflammatory disorders ,185. Extimmunity . Notablyimmunity . LOX1 waimmunity . exHSP90immunity  for promimmunity  and tumoimmunity . It is kimmunity ,195,196,immunity . CollectMolecular chaperones exert a myriad of functions in cells, including protein folding, degradation and transport and protecting proteins from denaturation or aggregation. However, their roles involved in immune stimulation, immune regulation and antigen presentation have attracted much attention. Growing evidence has indicated that cellular metabolism can dictate cell fate and govern the effector function of immune cells via multiple mechanisms including modulating epigenetic networks. It is especially true in fatty acid and amino acid metabolism, where metabolic byproducts fuel epigenetic regulation and transcriptional programs involved in immune cell metabolic reprogramming . The mit


PYL family genes in the apple . In this study, we identified 13 apple PYLs, termed MdPYL1-13, which could be classified into four groups according to structural features of the amino acid sequence. The gene structures and conserved motifs analysis found that the majority of MdPYLs had a similar number of exons and similar conserved motif profile in the same group. In addition, 11 gene pairs were identified to exhibit synteny by synteny analysis between the apple and Arabidopsis. Furthermore, we investigated MdPYLs transcript level in various organs of the red-fleshed apple  Langenf) ‘Xinjiang No.1’. The results suggested all MdPYLs within group I were expressed at relatively higher levels in all of the organs tested. However, the genes of group IV had little or no variation. Additionally, we found various hormone and stress-related cis-elements in the promoters of MdPYLs by analyzing cis-elements. Therefore, the expression levels of all MdPYLs were further detected under ABA, PEG, salt, and cold stresses in ‘Xinjiang No.1’ seedlings. We found that all MdPYLs except for MdPYL11 were upregulated by ABA treatment, 10 genes were upregulated by PEG treatment, 12 genes were upregulated by NaCl treatment, and six genes were upregulated by cold treatment (4 °C) while seven genes were downregulated. Thus, these MdPYLs might be involved in the defense against abiotic stresses. In addition, the interaction between 13 MdPYLs and two 2C protein phosphatases in the apple (MdPP2C65 and MdPP2C72) was investigated in yeast two-hybrid assays. These results suggested that MdPYLs may bind to MdPP2C65 and MdPP2C72 in different manners and with different intensity. Our studies provide useful information for further investigating and researching the regulatory mechanisms of PYL family genes in response to abiotic stresses in the apple.The pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) (known as PYLs for short) have been identified and characterized as the ABA receptors in some plants. However, little is known about the details regarding  Abscisic acid (ABA) is an important phytohormone involved in plant growth, plant development, and protection of plants against abiotic stresses ,2. The cArabidopsis genome [AtPYR1  and 13 AtPYLs  [PYLs, such as AtPYR1, AtPYL1, AtPYL2, and AtPYL3 inhibited PP2Cs in an ABA-dependent manner, while others including AtPYL4, AtPYL5, AtPYL6, AtPYL8, AtPYL9, AtPYL10, and AtPYL13 showed suppression of PP2Cs in the absence of ABA [PYLs, as ABA receptors, belong to the StAR-related lipid-transfer (START) superfamily containing a ligand-binding pocket enclosed by four conserved loops, CL1-CL4 , which is genome ,11, inclike1-13) . Some PYe of ABA ,13. In ae of ABA , droughte of ABA , and rooe of ABA ,17,18. PYL family in Arabidopsis, orthologous genes of PYL in other crops have been characterized at genome-wide levels, including six PYLs in sweet orange [PYLs in grape [PYLs in rice [PYLs in tomato [PYLs in rubber tree [PYLs in Brassica rapa [PYLs in cotton [PYLs in tobacco [PYLs in other plant species besides Arabidopsis has begun to be reported, and the various function of the PYLs in plant development and stress resistance was emphasized. For instance, overexpression of rice OsPYL3, cotton GhPYL10/12/26, or maize ZmPYL3/9/10/13 dramatically enhanced the sensitivity to ABA in transgenic Arabidopsis [OsPYL/RCAR5 or OsPYL3/5/9/11 also increased the sensitivity to ABA and the tolerance to drought stress in transgenic rice [PYL9 significantly enhanced the tolerance to drought stress and leaf senescence induced by drought in transgenic Arabidopsis and rice plants [Arabidopsis AtPYL4 (A194T), AtPYL5, maize ZmPYL8/9/12, or cotton GhPYL9-11A greatly improved tolerance to drought in transgenic Arabidopsis plants, respectively [PYLs in woody and lianas plants has also been reported. Overexpression of Populus trichocarpa PtPYRL1 or PtPYRL5 in both Arabidopsis and poplar also enhanced ABA sensitivity and drought resistance [VvPYL1 could be combined with ABA and repress the ABI1 phosphatase activity [VvPYL1 (VvRCAR7) exhibited high expression level in grape leaves treated by drought, salt, and cold stresses [Since the discovery of the t orange , eight Pin grape , 13 PYLs in rice , 14 PYLsn tomato , 14 PYLsber tree , 24 PYLsn cotton , and 29  tobacco . Recentlbidopsis ,28,29. Snic rice ,31,32. Oe plants . Moreoveectively ,34. In rsistance . In grapactivity . The VvPstresses . Arabidopsis and other plants such as maize, rice, and grape, the information concerning PYLs remains elusive in the apple. In this study, for the first time, we systematically identified the MdPYLs at the genome level in the M. domestica and performed their exon–intron structure and synteny analysis. Moreover, we investigated the spatiotemporal expression patterns of MdPYLs in various apple organs. Finally, we also examined expression patterns of MdPYLs under various abiotic stresses treatments. Our results will provide some clues for further investigating the function of PYL genes in apple tree growth and development, and resistance to abiotic stress. Unfortunately, in spite of an increase in reports in MdPYLs were present in the M. domestica genome through BLASTP by using 14 AtPYL sequences as references. Then, we termed MdPYL1 to MdPYL13 based on their chromosomal order  to 3252 bp (MdPYL1), with one to four exons in each sequence. However, the lengths of CDS and the corresponding MdPYL proteins both had no significant difference, varying from 555 bp (MdPYL1 and MdPYL7) to 777 bp (MdPYL4) and from 184 aa (MdPYL1 and MdPYL7) to 258 aa (MdPYL4), respectively. The predicted MW and pI values of these proteins ranged respectively from 20.13 kDa (MdPYL5) to 27.66 kDa (MdPYL4) and 5.02 (MdPYL10) to 8.30 (MdPYL11)  .MdPYLs were distributed on nine chromosomes including chromosome 1, 4, 5, 6, 7, 8, 12, 15, and 16  than monocotyledonous angiosperms (O. sativa and B. distachyon) (PYLs classifying into the same groups might have similar functions.We constructed a phylogenetic tree based on 109 protein sequences of hirsutum  via MEGAoup I–IV . Howevertachyon) . The resMdPYLs gene family, we carried out the analysis of amino acid alignment, phylogenetic tree, exon–intron structures, the conserved motifs, and segmental duplications , except MdPYL4 and MdPYL11 both contained two exons   .Arabidopsis genomes found the syntenies were clear and included the following ortholog pairs: MdPYL1, 7-AtPYL9; MdPYL2, 8-AtPYL5, 6; MdPYL5-AtPYL13; MdPYL6, 13-AtPYL1; MdPYL11-AtPYL4 and MdPYL12-AtPYL2 , defense and stress-responsive elements (TC-rich repeats), dehydration reaction elements (MYC), low-temperature-responsive elements (LTR), abscisic acid-responsive elements (ABRE), salicylic acid-responsive elements (TCA-element), ethylene-responsive elements (ERE), and MeJA-responsive elements   (MdPYLs with different cis-elements may differ in response to various abiotic stresses.Previous works have indicated that many software . In totaG-motif) . All MdPelements . For exa-element . The MdPelement) . The resMdPYLs in apple development, we detected their expression levels in different tissues and organs from Chinese wild apple clone ‘Xinjiang No.1’ using qRT-PCR. In general, most MdPYLs were expressed differently in various organs, showing they might have different functions (MdPYL5 (group IV) had little or almost no expression in the organs tested. In contrast, the expression levels of all MdPYLs within group I, including MdPYL1, MdPYL3, MdPYL7, and MdPYL9, were higher in all of the organs tested, and were more than 20 times of that in group IV. Furthermore, MdPYLs of group II, including MdPYL2, MdPYL4, MdPYL8, and MdPYL11, were preferentially expressed in the root and their expression level were more than fivefold of the other organs. At the same time, we found MdPYLs within group III expression exhibited the relatively higher levels than MdPYLs of group II in all tissue analyzed except for root. For example, the expression of MdPYL10 and MdPYL12 in peel, sarcocarp, and young fruit were over fivefold of all genes within group II  by carrying out qRT-PCR. Genes with expression levels that were increased or decreased more than two-fold, were used for the following analyses  reached the peak at 12 h after ABA treatment, while three genes  reached the peak at 24 h. Notably, MdPYL1, MdPYL2, MdPYL3, MdPYL4, MdPYL6, MdPYL8, and MdPYL13 all were upregulated more than fourfold after 12 h or 24 treatment, respectively. Similarly, following with PEG treatment, 10 genes  were sharply upregulated in expression after 6 h of treatment and reached the maximum at 12 h, while the expression of remaining three apple MdPYLs had no obvious changes  at 12 h after PEG treatment. After NaCl treatment, the expression of all MdPYLs except for MdPYL5 showed significant increases and surged to the peak at 6 h (more than threefold of CK)  . Followiegulated . PYLs can interact with PP2Cs to form PYL-ABA-PP2C triple complexes promoting the ABA signals transmission . HoweverPYLs encoding an ABA receptor family contained a similar ligand-binding pocket embraced by four conserved surface loops (CL1–CL4). In the study, 13 MdPYLs shared the typical structure were identified in the apple, with an almost equal number of AtPYLs from Arabidopsis  were positioned in syntenic regions of the apple and Arabidopsis genomes  were formed by segment duplication events in the apple genome  had high levels of expression in root, and 1g095700 showed predominant expression in leaf, 12g055990 and 8g082180 in fruit tissues [VyPYLs genes, especially VyPYL1, VyPYL4a, and VyPYL7-9 were expressed relatively strongly in roots, stems, and tendrils [PYLs play an important role in plant growth and development. The function of PYLs in Arabidopsis has been studied to support this idea. For example, overexpression of RCAR11, RCAR12, RCAR13, or RCAR14 inhibited the germination and root growth of transgenic Arabidopsis in an ABA-treated condition [AtPYL9 promoted ABA-induced leaf senescence in transgenic Arabidopsis [AtPYL8 promoted lateral root growth by interacting with AtMYB77 and enhancing its transcriptional activity [MdPYLs expression patterns and functions are still unclear in apple. Therefore, we speculated the roles of the apple MdPYLs based on qRT-PCR results in different tissues, and the information known of Arabidopsis counterparts  also increased drought resistance and accelerated stress-responsive gene expression in transgenic Arabidopsis [Arabidopsis, the functions of orthologous PYL proteins’ responses to stresses were reported in several plants, such as rice, cotton, tomato, and grape. In rice, ectopic expression of OsPYL3 improved cold and drought tolerance in transgenic Arabidopsis [OsPYL/RCAR5 increased drought and salt stress tolerance in rice [Gossypium, the expression level of many GhPYLs were downregulated by ABA treatment and upregulated by osmotic stress [GhPYL9-11A, GhPYL10, GhPYL12, and GhPYL26 improved the tolerance to drought stress in transgenic Arabidopsis [ABA receptors  and Vitis yeshanensis VyPYL9 all enhanced transgenic Arabidopsis drought resistance [MdPYLs to various abiotic stress conditions in the study (MdPYLs (except MdPYL11), 12 MdPYLs (except MdPYL5), and 10 MdPYLs  had significant expression changes when treated with ABA, salinity, and PEG, respectively  (MdPYL6 and MdPYL13 both contained low-temperature-responsive elements (LTR)  . Meanwhits (LTR) . This redPYL6/13 , was repand rice ,42. Fagus sylvatica FsPP2C1 in an ABA-independent manner and positively regulates ABA signaling during abiotic stress responses [Arabidopsis resistance to drought by inhibited HAB1 phosphatase activity in an ABA-dependent manner [MdPYLs. In conclusion, it seems that PYLs have various functions in the apple and play a key role in abiotic stress induced by ABA molecular signals.Furthermore, many evidences have shown that PYLs involve in plant response to abiotic stress by inhibiting PP2Cs in an ABA-dependent manner ,5,6,48. AtPYLs from the Arabidopsis genome to search for MdPYLs of the apple genome. The BLAST program was set to the default value  [MdPYLs on the chromosome were mapped by using the Gene Structure Display Server.We used the protein sequence of 14  < e−10) . Manual M. domestica, A. thaliana, B. distachyon, O. sativa, R. communis, T. cacao, V. vinifera, and G. hirsutum. Multiple alignments of protein sequences were analyzed by the neighbor-join (NJ) algorithm from 1000 repeated by MEGA 5.2 software [The phylogenetic tree was constructed using the PYLs amino acid sequences from software .MdPYLs was obtained using the Circos program [The homologous genes were searched by MCScanx software , and the program .The MdPYLs protein sequences were found from the apple genome, and their intron–exon structures were analyzed by GSDS . The MdPMdPYLs (The cis-acting elements were detected using the PLACE database in the 1500 base pair (bp) upstream of the gene initiation codon (ATG) of the MdPYLs .Malus sieversii f. Neidzwetzkyana (Dieck) Langenf) ‘Xinjiang No.1’, a wild apple resource with resistance to abiotic stress, was from Xinjiang of China.The red-fleshed apple (−1 indole-3-butytric acid (IBA) and 0.7 mmol L−1 6-benzylaminopurine (6-BA). After 30 days of growth, the ‘Xinjiang No.1’ seedlings were transferred to the medium, which supplied with 10% polyethylene glycol (PEG) 6000 or 100 mmol L−1 abscisic acid (ABA) or 100 mmol L−1 NaCl to induce different abiotic stress, respectively. Furthermore, the temperature for apple tissue culture seedlings cultured in a specific medium was adjusted to 4 °C to induce cold stress. The stress-treated seedlings were harvested at 0, 6, 12, and 24 h, then immediately frozen with liquid nitrogen and stored in a −80 °C freezer for follow-up studies.The apple ‘Xinjiang No.1’ tissue culture seedlings used for stress treatment was cultured in a specific medium with Murashigeand Skoog (MS) medium, 0.8% agar, 0.5 mmol LThe different apple organs, including roots, stems, leaves, pedicels, calyxes, anthers, filaments, receptacles, petals, pistils, sarcocarps, peels, young fruits (20 days after flowering), and seeds were collected from seven-year old ‘Xinjiang No.1’, which was obtained from tissue culture seedlings transplanted to the Jiao Zhou Experimental Farm  of Qing Dao Agricultural University.® 480 real-time PCR instrument . The PCR program was as followed: 95 °C for 2 min, 40 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s with a final dissociation stage. Actin of apple was used as an internal control for normalized gene expression levels. Repeat at least three times for each sample. Gene relative expression levels were calculated using GraphPad.Prism.5.0 software.RNA was extracted from the collected samples using the EASYspin Plant RNA Rapid Extraction Kit . The RNA concentration was measured with a NanoDrop 2000  instrument. The cDNA was obtained according to a reverse transcription kit . Specific primers were designed on NCBI-BLAST . AccordiFull-length sequences of 13 MdPYLs and two MdPP2Cs (MdPP2C72: XM_008359137.2 and MdPP2C65: XM_008371984.3) were respectively amplified by PCR from ‘Xinjiang No.1’ leaf using the specific primers with enzyme digestion sites . The ful−1, 10−2, and 10−3 were spread on plates containing SD/-Leu/-Trp, SD/-His/-Leu/-Trp/X-α-Gal medium with 10 mM 3-AT  and SD/-His/-Leu/-Trp/X-α-Gal medium with 10 mM 3-AT  and 10 μM ABA. The plates were photographed after four days of incubation at 30 °C. The experiments were repeated three times.After co-transformation into the Y2H Gold yeast strain, the yeasts were plated on a SD/-Leu/-Trp media. Then, the positive yeasts were grown in liquid SD-Leu/Trp medium for 12 h at 30 °C. Finally, 3 μL of the co-transformed yeast cells dilutions of 10MdPYLs were identified in M. domestica genome. Phylogenetic reconstruction and gene structure analysis demonstrated that MdPYLs could be divided into four groups, and that they had similar gene structures and high conserved motifs in the same group. Moreover, comparative genomic analysis showed that homologs of nine MdPYLs were located in corresponding syntenic regions of Arabidopsis. The MdPYLs expression analysis in various organs revealed distinct spatiotemporal patterns. Furthermore, gene expression analysis showed that MdPYLs are possibly involved in multiple abiotic stress responses . The results presented here call for further research aiming at revealing the potential important functions of these genes in the apple.Thirteen


As the spectrum of diseases keeps changing and life pace keeps going faster, the probability and frequency of diseases caused by human inflammatory reactions also keep increasing. How to develop effective anti-inflammatory drugs has become the hotspot of researches. It has been found that alkaloids from Chinese medical herbs have anti-inflammatory, analgesic, antitumor, anticonvulsant, diuretic, and antiarrhythmic effects, among which the anti-inflammatory effect is very prominent and commonly used in the treatment of rheumatoid arthritis, ankylosing spondylitis, and other rheumatic immune diseases, but its mechanism of action has not been well explained. Based on this, this paper will classify alkaloids according to structural types and review the plant sources, applicable diseases, and anti-inflammatory mechanisms of 16 kinds of alkaloids commonly used in clinical treatment, such as berberine, tetrandrine, and stephanine, with the aim of providing a reference for drug researches and clinical applications. Tripterygium wilfordii and Begonia kunmingshanensis of Celastraceae; Sinomenium acutum, Stephania tetrandra S. Moore, and Menispermum dauricum DC. of Menispermaceae [Coptis chinensis Franch., Aconitum carmichaelii, Aconitum kusnezoffii Reichb., Aconitum carmichaelii Debx., and Clematis chinensis Osbeck of Ranunculaceae; Gentiana macrophylla Pall. and Gentiana scabra Bunge. of Gentianaceae, and so on [Inflammation has been considered as the main risk factor of rheumatic immune diseases. As the incidence rate of this disease keeps increasing in recent years, how to effectively resist inflammation has attracted widespread attentions. The application of anti-inflammatory Chinese medical herbs not only enjoys a long history, a wide range of sources, and rich varieties, but also has complex and diverse pharmacological effects. Therefore, it has become a research hotspot to search for anti-inflammatory active ingredients in Chinese medical herbs, among which alkaloids are of great representativeness. Alkaloids are nitrogen-containing organic compounds with an alkali like properties. They are widely distributed in ermaceae ; Coptis nd so on . Some alnd so on . The folIsoquinoline alkaloids are widely distributed in 27 sections of plants, such as Menispermaceae, Berberidaceae, Papaveraceae, and Ranunculaceae . IsoquinCoptis chinensis Franch., the bark of Phellodendron chinensis Schneid., and Daemonorops margaritae (Hance) Becc. [Berberine, a kind of benzylisoquinoline alkaloid extracted from the rhizome of e) Becc. , has thee) Becc. . 8 mg/kgγ, interleukin-1 (IL-1), tumor necrosis factor (TNF-α), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) [Intraperitoneal injection of 30 mg/kg of berberine for 8 days can inhibit the increased prostaglandin E2 (PGE2) in paw swelling mice induced by formalin . The mec (MCP-1) . Berberi (MCP-1) .κB) and the transcription of inflammatory cytokines in ulcerative colitis mice induced by dextran sodium sulfate [α (TNF-α) but also the adhesion of polymorphonuclear leukocytes and vascular endothelial cells activated by TNF-α, so as to reduce the infiltration of inflammatory cells and lesions of colonic mucosa [In terms of diseases in the digestive system, berberine can protect the gastric mucosa from chemical factors. Berberine can inhibit the activation of nuclear factor kappa-B (NF- sulfate . Berberic mucosa .Stephania tetrandra S. Moore. and Stephania japonica (Thumb.) Miers [κB/COX-2 signaling pathway, but also inhibits histamine, platelet-derived growth factor (PDGF), IL-1β, and TNF-α in inflammatory reactions and promotes the expression of the anti-inflammatory cytokine IL-10 [Tetrandrine is a kind of bisbenzylisoquinoline alkaloid extracted from the root of .) Miers . It has .) Miers . Intrape.) Miers . The eff.) Miers . Tetrandne IL-10 .κB, thus inhibiting the production of IL-6, IL-8, and TNF-α inflammatory factors, as well as reducing the inflammatory response of pancreas [Intragastric administration of 6.25 mg/kg of tetrandrine can reduce the concentration of nitric oxide in serum and pancreatic tissue of rats with acute hemorrhagic necrotizing pancreatitis, reduce the activity of phospholipase A2, and inhibit the activation of NF-pancreas .κB inhibitor α, but also reduce the production of nitric oxide (NO) and prostaglandin E2 (PGE2), and the expression of matrix metalloproteinase-3 (MMP-3) and tissue metalloproteinase inhibitor-1 (TIMP-1) [In the inflammatory model of RAW264.7 cells induced by lipopolysaccharide (LPS), tetrandrine can not only inhibit the phosphorylation of NF-(TIMP-1) .Menispermum dauricum DC [Dauricine, a kind of benzylisoquinoline alkaloid extracted from the rhizome and rattan of ricum DC , has the4. Its mechanism is related to inhibiting the expression of TGF β1 and TNF-α [Dauricine can significantly improve neurological deficit symptoms and reduce the apoptosis rate of neurons in ischemic brain tissue . Moreovend TNF-α .β, IL-6, TNF-α, and intercellular adhesion molecule-1 (ICAM-1), but also the activity of inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), and cyclooxygenase-2 (COX-2) in LPS-stimulated macrophages [In vitro studies, it is identified that pretreatment of dauricine can dose-dependently inhibit not only the contents of NO, IL-1rophages .Intraperitoneal injection of 50 mg/kg of dauricine, with pharmacological actions similar to hydrocortisone, can inhibit the increased capillary permeability caused by histamine and acetic acid, inflammatory edema caused by xylene in mice, and edema of hind paws caused by carrageenan in rats . DauriciSinomenium acutum (Thunb.) Rehd. et Wils. [Sinomenine, a kind of benzyl alkaloid, is extracted from the stem and root of et Wils. . It can et Wils. .α, and IL-6 [Oral administration of 30 mg/kg of sinomenine can not only inhibit the activity of iNOS and COX-2 in rats but also inhibit the synthesis and release of PGE2 and leukotriene in inflammatory areas. Sinomenine can alleviate LPS-induced lung inflammation by inhibiting the expression of nitric oxide (NO), myeloperoxidase (MPO), TNF-κB and JNK signaling pathways. The mechanism is that it inhibits the expression of TNF-α mRNA and IL-1β mRNA in synovial cells by inhibiting NF-κB activity [Sinomenine achieves the anti-inflammatory effect by inhibiting NF-activity . Sinomenactivity . Sinomenactivity . Moreoveactivity .Lycoris radiata (L′ Her.) Herb., has a therapeutic effect on osteoarthritis, deformable osteitis, gout, ankylosing spondylitis, and other rheumatic autoimmune diseases. Intravenous injection of 3 mg/kg of lycorine has a therapeutic effect on formalin arthritis in rabbits and protein arthritis in rats [Lycorine, a kind of pyrrolidine alkaloid extracted from the bulb of  in rats . In the  in rats .μm) can inhibit activities of iNOS and cyclooxygenase-2 induced by LPS. The inhibitory effect is stronger than that of dexamethasone. In addition, lycorine can also inhibit the production of PGE2 and IL-6 in macrophages to play an antipyretic and analgesic role [Tolerance ability of inflammatory factors in mice gets improved after injecting lycorine. It is found that lycorine (5 sic role .μM) inhibited the activation of STAT1 and STAT3 stimulated by LPS [A STAT signaling pathway is one of the common pathways of physiological and pathological reactions in the human body. JAK-STAT  pathway gets involved in the signal transduction and regulation of many important inflammatory/anti-inflammatory cytokines . STATs ad by LPS .Menispermum japonicum Thunb. [α, IL-1β, and IL-6 in the mammary gland tissue of mastitis rats induced by LPS [κB degradation, but also improve the pathological changes of breast tissue. Intraperitoneal injection of 20 mg/kg of stephanine can reduce the expression of NF-κB, phospho-p38 MAPK, phospho-JNK, NLRP3, and IL-1β in the brain tissue of mice with focal cerebral ischemia, so as to play an anti-inflammatory role [β, and IL-18 but also increase the content of IL-12 in peripheral blood and nasal lavage fluid [Stephanine, a kind of simple isoquinoline alkaloid, is extracted from the root and tuber of m Thunb. . It has d by LPS . Stephanory role . Intragage fluid .Sophora flavescens Ait., Diphasiastrum veitchii., Piper nigrum L., Lobelia chinensis Lour., etc. [Piperidine alkaloids are derived from lysine, including piperidine, indolicidin, and quinolizidine. These alkaloids can be found in r., etc. . Piperidr., etc. .Sophora alopecuroides, has therapeutic effects on myocardial ischemia-reperfusion injury, acute renal injury, asthma, ventricular hypertrophy, pulmonary hypertension, and other cardiovascular diseases [α, and IL-1β in H9c2 cardiomyocytes decreases significantly after intragastric administration of 50 mg/L of aloperine for 48 hours, which suggests that aloperine can alleviate myocardial ischemia-reperfusion injury by inhibiting inflammatory response [β, TNF-α, NO, MCP-1, ADMA, ICAM-1, and VCAM-1, thus inhibiting the myocardial fibrosis and ventricular hypertrophy caused by isoproterenol [Aloperine, extracted from diseases . The conresponse . Intragaoterenol .β1 play an important role in the development of pulmonary hypertension. Pulmonary hypertension is often accompanied by a large number of inflammatory cells infiltration into pulmonary vascular lesions. The expression levels of NF-κB, TNF-α, and IL-1β protein significantly reduce after intragastric administration of 100 mg/kg of aloperine [κ, Bp65, p-IKKα, p-IκBα, TNF-α, and CyclinE1 [κB p65 signaling pathway [IL-6 and TGF-loperine . The resCyclinE1 . After t pathway .κB inflammatory signal pathway and the reduction of TNF-α and IL-1β levels [In terms of respiratory diseases, intragastric administration of 40 mg/kg of aloperine can improve lung function, alleviate asthma symptoms, and inhibit inflammatory responses in asthmatic mice. The mechanism is related to the regulation of the NF-β levels .Sophora flavescens, has therapeutic effects on rheumatoid arthritis and ankylosing spondylitis, as well as organ damage caused by endotoxin and exogenous toxicant [α, and NO [α and other inflammatory cytokines [Sophoridine, extracted from the roots of toxicant . Intragatoxicant . Intrapetoxicant . Its ant, and NO . Continu, and NO . The damytokines .Sophora flavescens, has therapeutic effects on rheumatoid arthritis, psoriasis, ulcerative colitis, ankylosing spondylitis, asthma, acute lung injury, acute respiratory distress syndrome, and cerebral infarction [Matrine, extracted from the root of farction .Hypodermic injection of 25 mg/kg of marine can inhibit the swelling of auricle caused by croton oil, the acute inflammatory swelling of the toes caused by carrageenan, or egg white . Moreoveα, IL-1β, and IL-6. Matrine can inhibit airway inflammation by inhibiting the signal transduction of NF-κB in airway epithelial cells of asthmatic mice, reducing the expression of SOCS3 as well as the production of reactive oxygen species (ROS) and inflammatory factors in alveolar macrophages [In terms of respiratory diseases, 3 hours after endotoxin injection, intraperitoneal injection of marine (10 mg/kg) can resist the pathological changes of lung tissue in mice caused by lipopolysaccharide and reduce pulmonary edema, pulmonary vascular leakage, and rise in temperature . Moreoverophages .α and can effectively reduce the secretion of IL-1α and IL-8 [Matrine also has a protective effect on lung injury caused by ulcerative colitis. The mechanism is to enhance the anti-inflammatory and scavenging oxygen free radical ability of lung tissue and upregulate the repair factors in lung and gut tissue by increasing the expression of ZO-1 and Occludin, which are closely related to the mucosa of lung tissue. Matrine has a strong inhibitory effect on TNF-and IL-8 .κB, TLR4, and TLR2 in ischemic brain tissue [In terms of nervous system diseases, injecting matrine (30mg/kg) into the jugular vein can inhibit the activity of 12/15-lipoxygenase and the expression of NF-n tissue . Moreoveμmol/L can significantly inhibit the expression of IL-6, IL-8, TNF-α, NF-κB, and intracellular adhesion molecule 1 in colon epithelial cells induced by lipopolysaccharide. Matrine at the dosage of 100 μmol/L can significantly resist the inflammatory damage of colon epithelial cells induced by lipopolysaccharide [In vitro, matrine at the dosage of 50 ccharide .Terpene alkaloids come from mevalonic acid. They can be classified into four categories, which include monoterpenes, sesquiterpenes, diterpenes, and triterpenes. Terpene alkaloids have antihypertensive, analgesic, anti-inflammatory, antipyretic, and sedative effects. Among them, gentianine, aconitine, and bulleyaconitine A have good anti-inflammatory effects .Gentiana scabra Bunge., the whole herb of Gentiana algida Pall., and the seeds from Trigonella foenum-graecum. Intraperitoneal injection of 90 mg/kg of gentianine for 10 days can reduce not only the increase of capillary permeability caused by egg white but also the formaldehyde-induced foot swelling in rats, accelerating the swelling to subside as well [Gentianine, a kind of monoterpene alkaloid, is extracted from the root of  as well . The eff as well . The conβ-induced inflammatory responses in rats' articular chondrocytes by inhibiting the activity of P38, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK). In addition, gentianine can also inhibit the release of matrix metalloproteinases (MMPs) induced by IL-1β and promote the expression of type II collagen [κb1, TNF-α, and IL-6). Oral administration of gentianine at the dosage of 60 mg/kg for 7 days has a therapeutic effect on contact dermatitis induced by 1-fluoro-2,4-dinitrofluorobenzene (DNFB) [α, IFN-α, IL-6, and MCP-1 in inflammatory tissues.Gentianine inhibits IL-1collagen . Oral ade (DNFB) . GentianAconitum carmichaeli Debx., has a therapeutic effect on rheumatoid arthritis, ankylosing spondylitis, and other autoimmune diseases [κB, reducing the contents of inflammation transcription factor NF-κB and inflammatory mediators TNF-α, IL-6, and IL-1β. Moreover, no evidence has been founded to support aconitine's toxicity to the cells of the lung tissue [Aconitine, a kind of diterpene alkaloids extracted from the root tuber of diseases . Oral addiseases . The effg tissue .Aconitum kusnezoffii Rchb. [γ, and PGE2 [α, and MCP-1 produced by LPS-induced RAW264.7 cells [κB1 and PKC-δ in the NF-κB signaling pathway in the lung tissues of asthmatic mice, but also reduce the contents of IL-4, IL-10, TNF-α, and MCP-1 in bronchoalveolar lavage fluid [Bulleyaconitine A, a kind of aconitine diterpenoid alkaloids, is extracted from the root tuber of ii Rchb. . Nowadayand PGE2 . Intrape.7 cells . Intrapege fluid .Purine alkaloids have the same purine ring structure and can be transformed into each other under specific conditions, mainly including caffeine, theophylline, theobromine, xanthine, and hypoxanthine. Among them, theophylline has a good anti-inflammatory effect and has been widely used in the clinical treatment of Chronic Obstructive Pulmonary Disease (COPD) and other pulmonary diseases.κB and the activation of inflammatory cells and T cells from peripheral blood to airway mucosa metastasis, thus playing an anti-inflammatory and immune regulatory role. Oral administration of 0.1 g of theophylline twice a day can reduce the contents of IL-4, IL-5, IL-6, IL-8, IL-17, TNF-α, and C-reactive protein (CRP) in COPD patients. Moreover, theophylline of this dosage can increase the activity and expression of histone deacetylase-2 in the blood of COPD patients and improve the sensitivity of the human body to glucocorticoids [Theophylline, extracted from tea and cocoa beans, has the functions of anti-inflammatory, bronchiectasis, immunity regulation, and improving the contractility of septal muscle. It has therapeutic effects on bronchial asthma, emphysema, and heart failure . Theophyrticoids .Moreover, a low dosage of theophylline can inhibit the abnormal expression of ET-1 induced by inflammatory cytokines in smooth muscle, which is one of its mechanisms of reducing the hyperresponsiveness of asthmatic airway.Organic amine alkaloid is a kind of important alkaloid. Nitrogen atoms in this alkaloid are not combined in a ring structure. Colchicine, betaine, ephedrine, demecolcine, and leonurine belong to this kind of alkaloids.Colchicum autumnale L., has been now widely used in the treatment of gout and the first choice for acute gout attacks. The 2016 European rheumatic union guidelines for the treatment of gout [Colchicine, extracted from the seeds and bulbs of  of gout  suggestsβ.Monosodium urate crystal deposition is the central link in the pathogenesis of gouty arthritis. When neutrophils phagocytize monosodium urate crystals, lysosomes can be induced to release a chemokine derived from glycopeptide crystals, which can significantly amplify the recruitment of neutrophils. Colchicine, by means of inhibiting the aggregation of neutrophils at the joints, weakens neutrophils' phagocytosis towards monosodium urate crystals and reduces the inflammatory response caused by partial neutrophils . Moreoveβ, and TNF-α in the aorta of mice exposed to tobacco for a long period of time could be reduced to achieve the purpose of vascular protection [In terms of heart disease, oral administration of 1.5 mg of colchicine for 5 days can reduce hypersensitive C-reactive protein (hs-CRP), IL-1, IL-6, IL-8, and Neutrophilic Alkaline Phosphatase 3 (NALP3) levels in patients' serum with acute myocardial infarction . Moreoveotection .Indole alkaloids, mainly derived from tryptophan, are the largest and most complex alkaloids. Indole alkaloids are divided into four groups: simple indoles, tryptophan indole, hemiterpenoid indoles, and monoterpene indoles.Uncaria rhynchophylla (Miq.) Miq. ex Havil. [μg/μL of rhynchophylline can inhibit the production of IL-1β and TNF-α in dopaminergic neurons and glial cells induced by lipopolysaccharide, so as to protect neurons. Rhynchophylline can also inhibit the phosphorylation of p38 MAPK, then block the nuclear translocation of NF-κB, and inhibit its transcriptional function, so as to inhibit the vascular endothelial cell damage caused by intermittent hypoxia [Rhynchophylline, a kind of monoterpene indole alkaloids, is the highest in the content of x Havil. . It has x Havil. . In vitr hypoxia .Strychnos nux-vomica Linn., has therapeutic effects on myasthenia gravis, rheumatoid arthritis, and hemiplegia caused by cerebrovascular diseases [Brucine, a kind of monoterpene indole alkaloids extracted from the mature seeds of diseases . Brucinediseases . In myasAs people learn more and more about nature, Chinese medical herbs have become important sources for the creation of new medicines. The purpose of researches on the anti-inflammatory effects and mechanisms of alkaloids in Chinese materia medica is to find new anti-inflammatory drugs with high selectivity and strong effect and improve the effectiveness, safety, and economy of the current clinical treatment in rheumatoid arthritis, ankylosing spondylitis, and other rheumatic immune diseases.There are many side effects of drugs currently used in the treatment of rheumatic immune diseases. Nonsteroidal anti-inflammatory drugs can increase the risk of gastrointestinal bleeding. Glucocorticoids can not only raise blood sugar and blood pressure but also aggravate osteoporosis. Immunosuppressive agents can give rise to fungal or atypical pathogen infections. Alkaloids in Chinese materia medica have unique advantages in the treatment of rheumatic immune diseases. They exert anti-inflammatory effects through multiple targets, without adverse reactions mentioned above. Their anti-inflammatory mechanisms include stimulating the pituitary adrenal cortex axis, promoting the release of adrenal cortex hormones, inhibiting the release of inflammatory mediators, interleukins, and tumor necrosis factors, and regulating the level of nitric oxide, the expression of cytokine mRNA, and so on .Anti-inflammatory alkaloids can be mainly divided into the following categories: isoquinoline alkaloids, indole alkaloids, pyridine alkaloids, terpenoid alkaloids, organic amine alkaloids, etc. This article summarizes anti-inflammatory mechanisms of alkaloids in Chinese materia medica, in order to provide a reference for screening active ingredients with anti-inflammatory effects and finding new therapeutic targets. However, the studies above are limited to animal experiments, and some of their mechanisms need further study. We will continue to explore the anti-inflammatory effects of alkaloids in Chinese materia medica in clinical practice and contribute our efforts to the development of new drugs with anti-inflammatory effects.