IL-17) family genes with susceptibility to human diseases, yet have obtained controversial outcomes. Therefore, we sought to update comprehensive synopsis of variants in the IL-17 family genes with susceptibility to human diseases.Genetic association studies have elucidated the link of variants in the interleukin 17  with susceptibility to 6 human diseases , moderate to 2 variants with risk of 5 diseases, weak to 5 variants with risk of 10 diseases. Bioinformatics analysis suggested that the variants with strong evidence might fall in putative functional regions. Additionally, positive relationships for 5 variants with risk of 4 diseases (based on two datasets) and 14 variants with risk of 21 diseases (based on one dataset) were considered noteworthy.Seven variants in IL-17 family genes are significantly linked with susceptibility to cervical, lung cancer, asthma, multiple sclerosis, rheumatoid arthritis and spondyloarthritis, and elucidates the crucial role of the IL-17 regions in the genetic predisposition to cancer or noncancerous diseases.This study offers updated and comprehensive clues that variants in the  IL-17), a homodimeric glycoprotein composed of 155 amino acids, remains a pro-inflammatory and its family genes contain six groups (IL-17A to F)   (Interleukin (IL) 17 (ophages) . It coulOVID-19) .IL-17F and found that rs763780 {His-to-Arg substitution at amino acid 161 (H161R)} in the third exon of the IL-17F gene influenced the susceptibility to asthma and chronic obstructive pulmonary disease (COPD) in the Japanese population   in pulation , 5. Sinc ovarian , gastric ovarian  and lung ovarian . In 2014l cancer .IL-17 family genes and the risk of diseases, the outcomes are controversial. In addition, an updated research synopsis with comprehensive functional annotation had not been conducted to assess the epidemiological evidence of associations with IL-17 family genes and risk of all human diseases thus far. Therefore, we carried out meta-analysis to elucidate the relationships of SNPs in the IL-17 genes with susceptibility to disease, offered the epidemiological evidence for variants with significant relationships, and evaluated the functions of significant variants using public sources.Even though previous studies evaluate the relationship between SNPs in Our research followed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement (PRISMA) and the Human Genome Epidemiology Network for systematic review of genetic association studies , 17.We screened genetic association studies from Pubmed and Web of science up to 30 Apr 2022 using “{interleukin-17} OR {IL-17} OR {IL-17}” AND “{variant} OR {variation} OR {polymorphism} OR {genotype} OR {single nucleotide polymorphism} OR {SNP}”. We also collected additional articles by retrieving published reviews, meta-analyses studies, etc.IL-17 family genes and susceptibility to human cancers or non-neoplastic diseases performed in case-control, cohort or cross-sectional studies (ii) they could provide the genotype data to calculate the odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) under additive genetic model, (iii) they were published in English by form of full-text. The exclusion criteria are presented, (i) they lacked sufficient information , (ii) the study was not focused on SNP in IL-17 family genes, (iii) they were not published as full reports, such as conference abstracts and letters to editors, (iv) they concentrated on cancer mortality.The inclusion criteria are as follows: (i) they were concentrated on the relationships between SNPs in Two authors extracted the data independently using a predesigned collection sheet and any disagreement could be solved with the corresponding author by discussion together. The extracted data were as follows: first author, publishing year, study design, country or region, ethnicity, gene name, variant, cases and controls, genotype counts, minor allelic frequency (MAF). When previous articles studied on the same or overlapping data, we only extracted data from papers with largest sample size and most detailed information.P < 0.05 was significant level unless otherwise stated, which were conducted using Stata, version 15 . Meta-analyses were performed for variants with at least three datasets. We used the Cochran’s Q test to evaluate the heterogeneity between studies . We analyzed the regions of promoter or enhancer activity, local histone modification, DNase I hypersensitivity, transcription factor binding motifs and proteins bound to these regulatory sites. In addition, we examined genome-wide cis-eQTL data in multiple tissues from two major eQTL databases: the Genotype-Tissue Expression Project  tool HaploReg (v4.1)  and the  Project  and the  Project  to deter Project .IL-17 family genes and 25 diseases (5 cancers as well as 20 noncancerous diseases). The distributions of SNPs (n) with human diseases were presented: asthma (n=5), autoimmune thyroid diseases (AITD) (n=1), cervical cancer (n=3), COPD (n=2), colorectal cancer (n=2), coronary artery disease (CAD) (n=2), functional dyspepsia (FD) (n=3), gastric cancer (n=4), hepatitis B Virus (HBV) infection (n=4), hepatocellular carcinoma (n=1), immune thrombocytopenia (ITP) (n=3), inflammatory bowel disease (IBD) (n=3), leprosy (n=2), lung cancer (n=4), multiple sclerosis (MS) (n=1), osteoarthritis (OA) (n=2), periodontitis (n=2), pre-eclampsia (PE) (n=2), psoriasis (n=1), recurrent miscarriage (RM) (n=1), rheumatoid arthritis (RA) (n=5), spondyloarthritis (SpA) (n=2), systemic lupus erythematosus (SLE) (n=1), tuberculosis (TB) (n=3), Type 1 diabetes mellitus (T1DM) (n=1).As presented in IL-17 family genes with 5 cancers as well as 20 non-cancer diseases based on at least 3 datasets, under an additive genetic model. As presented in P<0.05). The cervical cancer susceptibility had positive association with minor allele of rs2275913 in Asians (OR=1.391), rs3748067 (OR=1.493) and rs763780 (OR=1.184). Apart from that, rs2275913 had an elevated susceptibility to colorectal cancer (OR=1.452); the positive association could be found in Asians and mixed populations , rather than in Caucasians. Apart from colorectal cancer, rs2275913 had an increased predisposition to gastric cancer (OR=1.281); the positive association could be found both in Asians and Caucasians ; this SNP could increase risk of lung cancer (OR=1.213). Moreover, rs8193037 could elevate lung carcinoma susceptibility (OR=2.129).we carried out meta-analyses to investigate correlations of 10 SNPs in For non-cancer disease, current results showed that significant relationships with asthma risk were found for rs1889570 in Asians (OR=1.230), rs2397084 (OR=3.220), rs763780 (OR=0.536) and rs8193036 (OR=0.833). In addition, rs763780 had an elevated susceptibility to AITD (OR=1.623) in Asians. For CAD, significant relationships were found for rs2275913 (OR=1.179). Additionally, rs3748067 could reduce risk of CAD in Asians (OR=0.723). For HBV infection, rs763780 had a decreased susceptibility to HBV infection (OR=0.852), especially among Asians (OR=0.826). Apart from that, rs763780 could decrease susceptibility to ITP (OR=0.486) and risk of IBD (OR=0.750) in Asians. Apart from that, rs763780 had a reduced susceptibility to leprosy (OR=0.364) and risk of MS (OR=0.687), respectively. Interestingly, current results showed that rs2275913 could increase risk of OA (OR=1.232), SpA (OR=1.401) and SLE (OR=1.160), whereas decrease susceptibility to RA (OR=0.862) and TB (OR=0.594); rs763780 could increase risk of OA (OR=1.344), psoriasis (OR=1.571), SpA (OR=3.684) and TB (OR=1.380), respectively.Additionally, 8 SNPs  had no association with susceptibility to 14 human diseases  in additive model. Of these, 3 SNPs had no association with 3 diseases  with at least 2,300 case and 2,300 controls. Also, we calculated the statistical power to confirm whether the bigger sample size confirming these relationships is required in next study (2I < 25%) was assigned to 3 variants with risk of 2 cancers and 6 noncancerous diseases (10 associations); moderate heterogeneity (25% ≤ 2I ≤ 50%) was assigned to 3 variants with risk of 1 cancer and 3 noncancerous diseases (4 associations); high heterogeneity (2I > 50%) was assigned to 4 variants with risk of 3 cancers and 9 noncancerous diseases (14 associations). Moreover, the results indicated that publication bias existed (p < 0.10) in associations for rs3748067 and rs2275913 with colorectal cancer risk. Apart from that, sensitivity analyses indicated that some significant summary ORs were lost, including rs8193036 in asthma and AITD, rs763780 in HBV infection (excess of significant findings); rs763780 in AITD and ITP , and in IBD (HWE), and in leprosy .As shown in IL-17F rs1889570, IL-17A rs2275913, IL-17F rs763780, IL-17A rs8193037) and 2 cancer  as well as 4 noncancerous diseases , moderate to 2 SNPs  and colorectal and gastric cancer as well as 3 noncancerous diseases , weak to 5 SNPs  and 1 cancer  as well as 9 noncancerous diseases .As shown in P < 0.2 for FPRP)  had significantly associated with susceptibility to 4 cancers  as well as 10 noncancerous diseases ; of these, 5 SNPs  and risk of 4 noncancerous diseases  were considered noteworthy . Moreover, IL-17A rs2275913 and IL-17A rs8193037 had associated with predisposition to lung cancer. We also found that rs2275913 is weak LD with rs8193037 in East Asians (r2 = 0.1) and is uncorrelated with rs8193037 in Europeans and Africans (r2< 0.05).As shown in IL-17 family genes and predisposition to human diseases for the first time, which offers precise results for the SNPs and provides more variants and diseases that never been investigated before. Our study included 227 papers with 73,509 cases and 93,253 controls and performed a meta-analysis using 135 papers with available information to assess relationships of 10 variants with susceptibility to 25 diseases (5 cancers as well as 20 non-cancer diseases); 7 SNPs had positively associated with 18 human disease predisposition. Our study used the Venice guidelines and FPRP tests to grade the cumulative evidence of significant relationships. At last, 4 SNPs were assigned to strong evidence with predisposition to 6 human diseases , moderate to 2 SNPs and 2 cancer as well as 3 noncancerous diseases, weak to 5 SNPs and 1 cancer as well as 9 noncancerous diseases. Moreover, we attempted to construct functional annotations for these 4 variants with strong evidence using data from the Encyclopedia of DNA Elements Project and other public databases and then uncovered that the SNPs with strong evidence might fall in several putative regulatory regions. In summary, this study provides updated evidence that SNPs in the IL-17 family genes had significant associations with predisposition to lung, cervical cancer, asthma, RA, SpA and MS.Our study performs a comprehensive research synopsis and meta-analysis, summarizes and updates the associations between SNPs in IL-17, a kind of proinflammatory cytokine, plays a crucial role in both innate and acquired immune responses  is located at chromosome 6p12.2, and the encoded protein is a proinflammatory cytokine produced by activated T cells, which might involve in the development of human diseases   was protective in RA individuals   and non-rcinoma) . Consistal level . IL-17A ividuals . IL-17A Gene ID: 05 is loc therapy , which i therapy . Moreove therapy  and III  therapy , 42, indIL-17F gene (Gene ID:112744) is located at chromosome 6p12.2, and the protein encoded by IL-17F gene is a cytokine activated by T cells. It could stimulate the production of other cytokines, such as IL-6, IL-8, and CSF2/GM_CSF , yielding significant relationships between 22 variants and the risk of 47 types of carcinoma. For example, our results found that some non-cancerous diseases including autoimmune diseases (such as autoimmune thyroid diseases), alopecia areata, and some type of autoimmune blistering diseases (such as bullous pemphigoid), which have presented in our supplementary Tables  with at least 2,300 cases and 2,300 controls, which presented over 80% statistical power to detect an OR of 1.15 for a variant with MAF 0.20 (Type 1 error 0.05). Further study less than current individuals on these 3 variants for these 3 diseases will not yield fruitful results (Some limitations apply to this research: (i) even though we conducted a comprehensive search to screen eligible papers, some articles may have been missed. Also, some malignancies and non-cancer diseases could not be completely assessed by meta-analysis owing to insufficient information ; (ii) only ethnicity was assessed by subgroup, other factors  might be required to confirm or refute the relationships with susceptibility to disease; (iii) the unreasonable data, like errors in genotype, could not be investigated, and (iv) moderate and weak evidence should be explained with caution.IL-17 family genes were graded as demonstrating strong association to 2 cancer and 4 non-cancer disease risk. Apart from that, these findings provide a foundation for further demonstrating the variations in the IL-17 family genes are positively linked with susceptibility to cervical cancer, lung cancer, asthma, MS, RA, SpA, and highlight that the variants in IL-17 family genes might become a valuable genetic tool to investigate the pharmacological targeting potential of IL-17 family genes. We should further understand its biological pathway and apply these clues to clinical practice and public health for risk assessment and management.In summary, this large-scale meta-analysis identified that 4 SNPs in the The original contributions presented in the study are included in the article/TL, LY, and HC designed this work. TL and LY integrated and analyzed the data. TL, LY, and HC wrote this manuscript. TL, LY, XL, CZ, CJ, ZY, CF, and HC finished the related Tables and Figures. TL, LY, and HC edited and revised the manuscript. All authors approved this manuscript.This study was supported by funding from the Chongqing Natural Science Foundation (grant No. cstc2020jcyj-msxmX0257).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The mortality rate was 12%, with death due to comorbidities in most cases. Most fatalities occurred in patients with arterial thrombosis. Pediatricians should be aware of this life-threatening possibility facing children with SARS-CoV-2 infection or its multisystemic inflammatory complication, who abruptly develop neurological or respiratory impairment. A prompt intensive care is essential to avoid severe sequelae or even exitus.COVID-19 and multisystem inflammatory syndrome in children (MIS-C) have been associated with a higher incidence of hypercoagulability and thromboembolic events (TEs), even in children, leading to relevant morbidity, and mortality. However, our understanding of such complications in childhood is limited. To better understand the incidence, clinical manifestations, risk factors, and management of COVID-19 and MIS-C-related TEs in children, a review of the current literature and a brief update on pathophysiology are given. Sixty-two studies, describing 138 patients with TEs associated with COVID-19 or MIS-C, were included. The overall number of TEs was 157, as 16 patients developed multiple TEs: venous TEs represented the majority (54%), followed by arterial thrombosis , and intracardiac thrombosis (ICT) (8%). Within the venous TEs group, pulmonary embolism (PE) was the most frequent, followed by deep venous thrombosis, central venous sinus thrombosis, and splanchnic venous thrombosis. Notably, 10 patients had multiple types of venous TEs, and four had both venous and arterial thrombosis including a newborn. Most of them  had at least one predisposing condition, being obesity the most frequent (21%), especially in patients with PE, followed by malignancy (9%). In 35% of cases, no data about the outcome were available About one-third of cases recovered, 12% improved at discharge or follow-up, and 6% had persistent neurological  Despite the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primarily causing a respiratory illness, it has been associated with hypercoagulability and thromboembolic events (TEs). TEs are frequent in hospitalized adults with severe novel coronavirus disease 2019 (COVID-19), resulting in high rates of disability and mortality . In addi®, MEDLINE®, and MEDLINE®-In Process for English-language studies published from January 2020 through June 2022. PubMed databases were searched combining the keywords “COVID-19” OR “SARS-CoV-2” OR “coronavirus” AND “MIS-C” OR “PIMS” AND “thrombosis” OR “thromboembolism” OR “stroke” OR “thromboembolic” OR “pulmonary embolism” AND any of the following: “child” OR “children” OR “pediatric” OR “infant” OR “adolescent.” Titles and abstracts in English were evaluated for eligibility. References of identified relevant articles were reviewed and papers from these sources were also included. Thereafter, a further selection was made including those articles in which one or more clinical data concerning patients with TEs could be obtained. The selected articles were reviewed by two independent authors (ST and CR) and judged on their relevant contribution to the study subject. Subsequently, according to the type of thrombosis, all TEs were categorized into arterial thrombosis , venous thromboembolisms (VTEs) , and ICT. Epidemiological and clinical findings, laboratory values, data on treatment, and outcome were reported. TEs were further distinguished as occurring in children with COVID-19, when the patient had positive real-time polymerase chain reaction or antigen SARS-CoV-2 tests on nasal or nasopharyngeal swabs, or in patients with MIS-C, defined according to the US Center for Disease Control and Prevention , 3 were case-series including ≥ 3 patients , 9, 10, These three subgroups had different gender distribution and clinical presentation. Considering the overall number of selected cases (138 patients), we found that the rate of symptomatic TEs was 72% (100/138), the rate of declared asymptomatic ones was 16% (22/138), and in 12% (16/138) the symptoms were not reported.Within the VTEs group, PEs were the most frequent events , followed by DVT , CVST , and SVT in 4 (3%). Notably, 10 patients had multiple types of VTEs and four had both venous and arterial thrombosis; among them, a newborn with a thrombus localized both in the right pulmonary artery and into the right ventricle was described  were females, with a median age of 13 years (IQR 11–16 years); 33 cases (85%) had COVID-19, and the remaining 6 had MIS-C (15%) . Their cDVTs were reported in 29 cases: 16 females  unspecified; the median age was 13 years (IQR 10–14). In 10 cases (34%), DVT developed in the context of MIS-C. DVTs were associated with PE in 7 cases, AIS in 2, CSVT and ICT in 1 case each, respectively.CVSTs were reported in 13 children; 9 (69%) were males, 4 (31%) were females, and the median age was 13 years IQR 7–15). In 2 patients, CSVT was associated with other TEs , and in 1 with AIS . Clinica. In 2 paSVTs were reported in 4 cases —all with COVID-19 —with a mThe total number of patients with ATs was 59, including 54 with AIS, 2 with splenic AT , 2 with The majority of patients with AIS were males , with a median age of 10 years (IQR 5–15); 34 children (63%) had COVID-19 and 20 (37%) had MIS-C . The mosICT was found in 13 patients (9%): 6 were females (46%), with a median age of 11 years (IQR: 4–12); 7 cases (54%) with COVID-19 and 6 (46%) with MIS-C. The clinical manifestations, reported in 9 cases, included hemodynamic compromise in 7 cases (54%), gastrointestinal symptoms in 3 (23%), and dyspnea with a decreased level of consciousness in 1 (8%). ICT was associated with PE in 3 children , 19, andThe presence of any risk factors correlated with TEs was reported in 130/138 patients . Most ofD-dimer values were reported in 86 cases (62%) and were elevated in most patients  . Among tTreatment was reported in 97 patients : 24 25%, 22. Twesequelae (mostly secondary to AIS), including hemiparesis in 3 children, facial palsy, dysarthria, and aphasia in 2, and weakness in 1. A neonate had abdominal aortic thrombosis requiring leg amputation  completely recovered, while 17 (12%) improved at discharge or at the last follow-up. Nine patients (6%) had persistent neurological putation . One patThe strong association between COVID-19 and coagulopathy suggests that multiple molecular pathways are involved and dysregulated through the disease progression, contributing to the development of thrombosis. First of all, endothelial dysfunction and barrier disruption lead to immune cell infiltration, and proinflammatory cytokine production, as well as thrombosis . In deteSevere COVID-19 is frequently burdened by life-threatening thrombotic complications in adults. Conversely, TEs are uncommon in children with COVID-19 and MIS-C. Nevertheless, both these conditions display a proinflammatory perturbation activating the coagulation system, which is emphasized in patients with a complicated clinical course. The earliest case of TE related to COVID-19 in childhood was reported in the first published MIS-C case series, in a 14-year-old boy who died after developing AIS with infarction of right anterior and middle cerebral arteries while on ECMO for acute multiorgan dysfunction, despite anticoagulation with UFH . Since tIn a recent multicenter study, the rate of thrombosis was described in a large cohort of patients < 21 years, hospitalized with SARS-CoV-2 infection or MIS-C, across 7 pediatric hospitals in the United States over 5 months. Overall, 16 patients with VTE, 3 with ICT, and 1 with stroke were described [5]. The rate of TEs in asymptomatic patients with SARS-CoV-2 (0.7%) was similar to that previously reported in hospitalized children (0.58%) . Conversely, in symptomatic patients, the rate of TEs was 2.1% in cases with acute COVID-19, 3.2% in those with MIS-C, and higher in those patients with a severe disease requiring ventilatory support and PICU admission. Data from a multi-institutional cohort study from the Tri-State Pediatric COVID-19 Research Consortium found a similar TE rate (4.3%) considering both patients with COVID-19 and MIS-C, but a lower rate (1.4%) in the patients with MIS-C .Analyzing data from the present review, we found that VTEs are the most frequent thrombotic complications, representing more than half of cases. Among them, PEs and DVTs are the most frequent sometimes with simultaneous localizations. Arterial thrombosis, found in more than one-third of cases, is mainly represented by AIS, while ICTs represent a smaller but relevant group (8%). TEs were mostly found in COVID-19 cases (68% vs. 32%), but this reasonably depends on the relatively low incidence of MIS-C compared with COVID-19. Furthermore, in the setting of MIS-C, ATs occurred more frequently than VTEs. The overall distribution of cases by sex was similar. However, considering different types of thrombosis, we observed that about two third of patients with ATs were males, whereas VTEs occurred more frequently in females (63%). The majority of cases were older than 11 years (62%). The central nervous system was the most commonly affected site, involved in 66 cases , followed by the lungs (PE reported in 39 patients). The heart was affected in 14 cases, mostly with ICT (13 patients), and coronary impairment in 1; the remaining cases had TEs localized at lower/upper limbs (29 cases with DVT) or visceral vessels (4 cases with SVT and 4 AT). Overall, stroke appears to be a rare, but severe complication of COVID-19. Both an International Pediatric Stroke Study survey of 61 international hospitals and a retrospective cohort study at seven US children’s hospitals suggested that the prevalence of stroke among children hospitalized for COVID-19 was < 1% , 26. TheAbout one-third of all the patients were previously healthy, while 57% had a history of one or more comorbidities. ECMO, required for severe cases, represents a medical procedure highly related to TEs development, as well as the presence of CVL, confirming the already well-known association between thrombosis and central venous catheters in children . These pMost patients with TEs received anticoagulant treatments. Heparin was used in more than half of the cases: LMWH in 30 cases, followed by UFH (12 cases), often given subsequently in the same patient; rarely other anticoagulants (apixaban), were reported. There is no evidence to support routine prophylactic therapy in patients with COVID-19 or MIS-C , and thiThis study represents a thorough review of the available English literature on a most relevant topic, TE complications in children with COVID-19 or MIS-C, and it provides an extensive understanding of the incidence, clinical manifestations, risk factors, and management of such complications in pediatric age. However, this review has some limitations. First of all, the collection of clinical cases and case series does not allow statistical analysis; therefore, no correlation between TEs and other clinical parameters could be established. Besides, thromboprophylaxis has not been assessed, as this information was not homogeneously reported (or even absent) in the selected articles. The relationship between thromboprophylaxis and TEs was out of the aims of this study and should be further investigated.ST and CR conceived the study and prepared the original draft. ST, CR, and FP performed the analysis and prepared tables and figures. DL, GI, MR, and GS critically revised the manuscript. All authors have read and agreed to the final version of the manuscript.


Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder caused by impaired B-cell function and antibody production. It commonly presents with chronic sinopulmonary and gastrointestinal manifestations. It is also associated with transformation to acute myeloid leukemia. However, the association of CVID with myelodysplastic syndrome (MDS) is rare. This case report aims to present one such rare association in a 26-year-old patient presenting with severe thrombocytopenia. Bone marrow biopsy revealed hypercellular marrow with 80-90% cellularity along with an increase in CD34 blasts. Cytogenetics revealed loss of the Y chromosome. Diagnosis of MDS with excess blasts-2 was confirmed with a Revised International Prognostic Scoring System score of 4, placing the patient in the intermediate-risk category. The patient was started on azacitidine, a hypomethylating agent. A referral to a bone marrow transplant was also done for the consideration of an allogeneic stem cell transplant. Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder caused by impaired B-cell function and antibody production. Although patients present with varied clinical presentations, the presence of chronic sinopulmonary and gastrointestinal manifestations are characteristic manifestations. It is also associated with autoimmune disorders (immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA)), inflammatory conditions , and malignancies  -3. An exA 26-year-old with a history of CVID since childhood, on treatment with intravenous (IV) immunoglobulin every three weeks, presented to a hematologist, with an abnormal blood picture Table .The patient’s platelet count had remained stable between 50,000 and 75,000 cells/μL between appointments. The patient also complained of 40 to 50-pound intentional weight loss.FMS-like tyrosine kinase-3 (FLT-3) mutation, commonly associated with acute myeloid leukemia (AML), was negative. Normal results were noted in chromosomes 5, 7, 8, 11, and 20 probe sets. A diagnosis of MDS with excessive blasts (MDS-EB) was made. No abnormalities were detected in the following genes: FLT3, IDH1, IDH2, and NPM. Next-generation sequencing revealed the following variants: IKZF1 N159S; KIT D816V; KRAS K117N; NF1 R711Pfs*37; NRAS G60V, G12D; and PHF6 R225.A bone marrow biopsy showed hypercellular bone marrow with 80-90% cellularity. An increase in CD34-positive blasts of about 15-16% of total cells was noted Figures . CytogenA computed tomography scan of the chest, abdomen, and pelvis revealed the progression of splenomegaly to 26 cm (compared to 19 cm in 2018). Thrombocytopenia was autoimmune, due to CVID, with further worsening secondary to MDS-EB. Our patient had a loss of chromosome Y, which is a very good prognostic marker. A revised International Prognostic Scoring System (IPSS-R) score of 4 placed the patient in the intermediate-risk category. IPSS-RA score of 2.68 places a patient in the low-risk category. According to the National Comprehensive Cancer Network (NCCN) guidelines, the patient was started on a hypomethylating agent, azacitidine. A referral to a bone marrow transplant was also done for the consideration of an allogeneic stem cell transplant. The patient passed away three months after initiation of the treatment due to infective endocarditis.TACI, BAFF-R, CD19, CD81, CD20, CD21, TWEAK, and NFKB2 [2 = 79.2%) [The association of MDS with CVID is quite unusual. CVID disorders are rare autosomal recessive disorders with a mean incidence of 0.676 ± 0.83 per 100,000 globally, with incidence increasing to 3.374 per 100,000 in developed nations . AlthougSF3B1, TET2, SRSF2, ASXL1, DNMT3A, RUNX1, U2AF1, TP53, and EZH2 [MDS is a hematological malignancy with ineffective hematopoiesis due to de novo mutation primarily in oncogenic genes. It most commonly occurs in individuals greater than 65 years of age, with a mean age of 77 and a male preponderance . Patientand EZH2 . MDS is ASXL1 and RUNX1, which were positive in previously reported cases, were negative in our case. However, KRAS mutation, which was positive in our case, has been reported in association with MDS, suggesting a possible underlying pathological mechanism [Investigations for the diagnosis of MDS include peripheral blood studies  and bone marrow studies  . Previouechanism . GATA2 dTreatment options range from a regular follow-up to low-intensity therapy  to high-intensity therapy (induction chemotherapy or hematopoietic stem cell transplantation (HSCT)) . TreatmeCVID is a rare primary immunodeficiency disorder caused by impaired B-cell function. Due to its more common association with autoimmune disorders (ITP and AIHA) and hematological malignancies (leukemia and non-Hodgkin lymphoma), abnormalities in common blood reports such as cytopenias have to be thoroughly investigated. With growing evidence for the presence of MDS in CVID patients, physicians should have a low threshold for suspicion of MDS. Peripheral blood smear and bone marrow aspirate findings help establish and categorize MDS. The cytogenetic analysis combined with molecular genetic testing aids in defining prognosis and treatment decision-making. Further studies are needed to find specific markers to identify MDS in CVID patients.


Multiple sclerosis is a progressive demyelinating central nervous system disorder with unknown etiology. The condition has heterogeneous presentations, including relapsing-remitting multiple sclerosis and secondary and primary progressive multiple sclerosis. The genetic and epigenetic mechanisms underlying these various forms of multiple sclerosis remain elusive. Many disease-modifying therapies approved for multiple sclerosis are broad-spectrum immunomodulatory drugs that reduce relapses but do not halt the disease progression or neuroaxonal damage. Some are also associated with many severe side effects, including fatalities. Improvements in disease-modifying treatments especially for primary progressive multiple sclerosis remain an unmet need. Several experimental animal models are available to decipher the mechanisms involved in multiple sclerosis. These models help us decipher the advantages and limitations of novel disease-modifying therapies for multiple sclerosis. More than 2.8 million people live with multiple sclerosis (MS) worldwide, and the prevalence has been increasing . The meaThe clinical manifestations of MS include temporary vision loss, sensory and motor problems, fatigue, impaired bowel and sexual functions, cognitive deficits, and paralysis –4. DistiHLA-A*02:01, HLA-DRB1*15:01, HLA-DRB5, HLA-C, and TNF, have been firmly associated with MS susceptibility  loci, such as tibility . In Afriin Japan . Interlein Japan . In addi+ T cells of patients with MS proliferated and secreted interferon- γ (IFN- γ), a characteristic of the Th1 subset, but not IL-17 when challenged with the myelin oligodendrocyte glycoprotein 35–55 (MOG35 − 55) peptide in vitro -infiltrating CD4in vitro , a propoin vitro . Increasin vitro . In addiin vitro . The intin vitro . Since tin vitro , it is unt in MS .+ T cells increased during relapse, which was not associated with EBV and HHV6 plasma viral loads. Although EBV infection has been hypothesized to contribute to MS development in the context of other predisposing conditions, such as the HLA genotype, vitamin D deficiency, smoking, and altered T-cell responses  were approved for MS treatment by the Food and Drug Administration (FDA) . Th effects , 45. Sev effects –50. Most effects , 27, 28. effects , dimethy effects , and nat effects , affect  effects , glatira effects  and dime effects . Nataliz effects . Systemi effects . However effects . Earlier effects . Since C effects . Other B effects  that wasSince 2018, several second-generation molecules with reduced gastrointestinal side effects have been approved for the treatment of MS by the FDA . DiroximSeveral other DMTs outnumbering those approved for MS treatment failed to meet the primary study endpoint and progress to a subsequent clinical trial because of commercial decisions. These include antibodies against the IL-12/23 p40 subunit , anti-CDin vitro. Significantly, they also did not improve the clinical outcome in patients with MS. Thus, effective methods of inducing antigen-specific tolerance in encephalitogenic T cells without causing adverse reactions remain an unmet need.In addition to these non-specific drug therapies, several attempts were made to induce antigen-specific tolerance in encephalitogenic T cells, which would ensure stable and adequate protection against autoimmune diseases without off-target effects . Th139 − 151 peptide or peptides derived from MBP exhibited relapsing-remitting EAE (RR-EAE) (b) mice, while causing atypical EAE characterized by ataxia, proprioception defects, and axial rotary clinical presentation in C3HeB/Fej (H-2k) mice , has been successfully induced in monkeys, guinea pigs, rats, and mice, following immunization with the whole-brain and spinal cord extracts and peptides derived from myelin proteins, such as MBP, PLP, and MOG [reviewed in Ref. ]. The mo(RR-EAE) , and thi2k) mice , 67. Aty2k) mice . In one 2k) mice , while o2k) mice . The bra2k) mice , 68. Sin2k) mice , atypica35 − 55 region proved to be an immunodominant epitope eliciting T- and B-cell responses and EAE in most strains of mice (35 − 55 was identified as an autoantigen in patients with MS (b) mice with the MOG35 − 55 peptide elicited EAE (35 − 55 peptide immunization induced a robust and long-lasting progressive EAE (PEAE) in non-obese diabetic (NOD) (H-2g7) mice  mice –77. Integ7) mice –72, but g7) mice –77, indi with MS  provided with MS , 69 .–80.35 −  with MS . Immuniz induces a disease similar to PPMS involving the brain, brainstem, and spinal cord . The TMEEAE models have traditionally been used to benchmark the efficacy of various disease-modifying therapies. However, several inconsistencies between mice and humans concerning the outcome of these attempts have been intensely debated –93. A fevia epigenetic mechanisms , and valproic acid corroborated with reduced CNS inflammation and demyelination in mice , 79, 80.ry cells , and redin vitro . Histonemulation , akin tomulation . By contnd Rorγt . Howevernd Rorγt  and the nd Rorγt , adminisnd Rorγt  providedmature (MHC class II+) CD11b+Ly-6G+ neutrophils and, to a lesser extent (MHC class II+) CD11b+Ly-6C+ mature monocytes in the peripheral lymphoid compartment before the onset of the peak clinical disease  without overt activation in vitro  has been proposed to be crucial for EAE , surprisg GM-CSF , promineg GM-CSF , transcrg GM-CSF , and RORg GM-CSF , were neg GM-CSF . Howeverg GM-CSF . The trag GM-CSF , similarg GM-CSF . TSA treNOD mice , 76. AltNOD mice , it is cNOD mice , 117. StNOD mice , while tt brains . Valproit brains . Collect35 − 55 upregulated the transcription of Hdac11 in the CNS, but none of the 11 Hdacs in the peripheral lymphoid cells  was upregulated in the CNS of TSA-treated mice . Thus, HAuanema freiburgensis with class I HDAC inhibitors butyrate and valproic acid, and the broad-spectrum HDAC inhibitor TSA increased the acetylation of histones 3 and 4 (Recent work has unraveled the inheritance of non-DNA sequence-based epigenetic information, epimutations, across several generations in yeast to humans . The sig 3 and 4 . Notably 3 and 4 , 129–132 3 and 4 –76, asth 3 and 4 , lupus ( 3 and 4 , 135, an 3 and 4 , in anim 3 and 4 , and selThis review discusses the effects of HDAC inhibitors on EAE regulation  and, by SJ conceived and executed the project and wrote the first draft of the manuscript. AJ conducted most of the experiments reported in this article and edited the manuscript. All authors contributed to the article and approved the submitted version.The work described in this article was supported by the University of Illinois at Chicago.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, heterosis-related grain weight QTLs and the basis of yield advantage among subspecies has not been well elucidated. We herein de novo assembled the chromosome level genomes of an indica/xian rice (Luohui 9) and a japonica/geng rice (RPY geng) and found that gene number differences and structural variations between these two genomes contribute to the differences in agronomic traits and also provide two different favorable allele pools to produce better derived recombinant inbred lines (RILs). In addition, we generated a high-generation (> F15) population of 272 RILs from the cross between Luohui 9 and RPY geng and two testcross hybrid populations derived from the crosses of RILs and two cytoplasmic male sterile lines . Based on three derived populations, we totally identified eight 1,000-grain weight (KGW) QTLs and eight KGW heterosis loci. Of QTLs, qKGW-6.1 and qKGW-8.1 were accepted as novel KGW QTLs that have not been reported previously. Interestingly, allele genotyping results revealed that heading date related gene (Ghd8) in qKGW-8.1 and qLH-KGW-8.1, can affect grain weight in RILs and rice core accessions and may also play an important role in grain weight heterosis. Our results provided two high-quality genomes and novel gene editing targets for grain weight for future rice yield improvement project.Following the “green revolution,”  In addivolution . As statvolution . Therefoution  or YTA (indica) were used to explore the heterosis loci of KGW. These results provided a new insight into the diversity mechanism of grain weight in rice.In this study, we performed the chromosome-level O. sativa ssp. indica/xian  and O. sativa ssp. japonica/geng  were planted in the field in Wuhan, China, in 2016, These two subspecies have many significant differences in important agronomic traits, including plant height, number of tillers, and heading date, for example, Luohui 9 has excellent agronomic traits, and RPY geng has an ideal plant architecture  and a 1% agarose gel electrophoresis, respectively. Libraries for Illumina short-read and single-molecule real-time (SMRT) sequencing  were prepared according to the respective manufacturer’s instructions. The short-read DNA libraries were sequenced by paired-end (2 × 150 bp) method on Illumina HiSeqTM 2,500  and the SMRT sequencing were sequenced on the PacBio RS II platform. In addition, Hi-C reads of DNA of young leaves from F1 progeny of Luohui 9 and RPY geng were sequenced on Illumina HiSeqTM 2,500 paired-end (2 × 150 bp) sequencing according to standard protocol. The total RNA of mixed tissues  was extracted for RNA-seq libraries following the manufacturer’s standard protocol. Then, RNA-seq libraries were sequenced on an Illumina HiSeqTM 2,500 paired-end (2 × 150 bp) and raw reads were filtered using Trimmomatic software as described previously .PacBio RSII sub-reads were filtered by the PacBio SMRT-Analysis package including removing the adapters, low quality, and short length reads  and sub-reads after filtering were corrected by Illumina reads using an error correction module embedded in Canu v1.5 . The higde novo, homolog-based, and transcriptome-based strategies. Augustus v2.4  databases including GO, KEGG, KOG, TrEMBL, and Nr databases using BLAST v2.2.31  were developed by crossing RILs (F14) with Z7A (japonica) and YTA (indica) in 2019LS. KGWs of Z7A-TCF1, YTA-TCF1, and RILs were investigated in 2019HN.A 272 RILs and their parents were planted in the Ezhou  Experimental Base of Wuhan University, Wuhan City, Hubei Province in April 2017 (2017EZ) and in April 2018 (2018EZ), the Hybrid Rice Experimental Base of Wuhan University in Lingshui City , Hainan Province in November 2019 (2019LS), and Breeding Experimental Base of Wuhan University Tianyuan Co., Ltd in Hannan District , Wuhan City, Hubei Province in May 2019 (2019HN). Two testcross populations  totally identified 39,255 and 39,440 gene models among RPY geng and Luohui 9 genomes, of which 96.81 and 94.75% had at least one annotation result in GO, KEGG, KOG, TrEMBL, or Nr database /1,212 (RPY geng) duplications between RPY geng and Luohui 9 involving 2,234 SV-related genes in Luohui 9 and 1,544 SV-related genes in RPY geng . Notablyjaponica/geng and indica/xian subspecies, respectively, yield traits of their F1 and many RILs showed obvious over-parent dominance. To resolve yield-related genes, we here conducted trait surveys and linkage analysis of KGW based on the previously constructed high-density genetic map  were only detected in one specific ecological environment, and were possibly environment-specific KGW QTLs.We totally identified eight KGW QTLs on Chr 2, Chr 3, Chr5, Chr6, Chr8, and Chr10 . Of QTLsqKGW-6.1 and qKGW-8.1, the remaining QTLs all had complete or partial overlap with Meta-QTLs, or contained the known KGW genes in these intervals  RILs of qKGW-6.1 showed greater KGW than Luohui 9 (BB) RILs (qKGW-8.1 showed the opposite result relative to qKGW-6.1 (qKGW-6.1 AA + qKGW-8.1 BB) had the largest KGW in all tested environments  RILs . InteresqKGW-6.1 . This suronments . These rqKGW-8.1 could be detected repeatedly in three environments with phenotypic interpretation rates of 5.11–9.96. However, no known genes directly related to grain weight were found in qKGW-8.1. We therefore traversed the 92 gene annotation results in qKGW-8.1 and tried to correspond them to the phenotypic differences between the parents. We found that Ghd8 is located within the qKGW-8.1 interval, a gene reported to be closely associated with heading date and yield  because Ghd8 is the only gene within block174811. In 532 rice core accessions, a functional snp (vg0804334484) was found in Ghd8 gene, containing A, C, and N alleles, and the N allele was eliminated in further phenotypic comparisons due to uncertainty about its base type. We found that different allele RILs or core accessions displayed significantly different KGW, suggesting that Ghd8 may play a role in KGW regulation   . Ghd8, ade novo assembled genomes of an indica rice (Luohui 9) and a japonica rice (RPY geng) at the chromosome level and analyzed the KGW trait of their derived RIL populations. We concluded that the substantial genetic diversity of KGW in RILs were closely related to genome variations and allele aggregation difference of KGW QTLs. Importantly, we identified two novel KGW-related QTLs (qKGW-6.1 and qKGW-8.1) and several KGW heterosis loci in three derived population. Based on the genotyping results in RILs and 532 rice core accessions, Ghd8 in qKGW-8.1 was presumed to play an important role in GW regulation.In the present study, we PRJCA010706.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ngdc, YL and WK conceived and designed the experiments. WK performed genome assembly, analyzed the data, prepared the figures and tables, and wrote the manuscript. XD conducted a field survey of agronomic traits, QTL mapping, and allele genotyping in RILs and rice core accessions. ZW participated in the construction of the recombinant inbred lines and revision of the manuscript. YW provided help in genome annotation. ZL performed repetitive sequence annotation of RPY geng and Luohui 9 genomes. MZ collected Meta-QTLs and known genes performed parts of figures and tables. All authors read and approved the final version of the manuscript.


The WGCM anode enhanced the maximum power density of MFCs by 167.9% compared with the carbon felt anode, while nano-TiO2/WGCM anode additionally increased the value by 45.8% to achieve 1396.2 mW/m2. WGCM enhancement was due to the 3D porous structure, the good conductivity and the surface hydrophilicity, which enhanced electroactive biofilm formation and anodic electron transfer. In addition, nano-TiO2 modification enhanced the enrichment of Acinetobacter, an electricigen, by 31.0% on the anode to further improve the power production. The results demonstrated that the nano-TiO2/WGCM was an effective anode for power enhancement in MFCs.A three-dimensional (3D) anode is essential for high-performance microbial fuel cells (MFCs). In this study, 3D porous carbon monoliths from a wax gourd (WGCM) were obtained by freeze-drying and carbonization. Nano-TiO Microbial fuel cells (MFCs) are an environmentally friendly biotechnology that uses electroactive bacteria (EAB) as anode catalysts to degrade organic matter in water and directly convert chemical energy into electrical energy, which has been widely studied in electricity production, wastewater treatment, biosensors, chemical synthesis, bioremediation, etc. ,2,3,4,5.2 nanoparticles could act as a mediator to enhance the electron transfer from the EAB to the electrode . Th. ThAcine2. WGCM and nano-TiO2/WGCM microporous structures were beneficial for EAB attachment and quick start-up. In addition, the output voltage and maximum power density of MFCs using WGCM and nano-TiO2/WGCM anodes were significantly enhanced compared with the CF control group, profiting from the 3D structure of WGCM, good hydrophilic surfaces and high conductivity. The excellent performance of the nano-TiO2/WGCM was due to the enrichment of the electrogen Acinetobacter, which further improved the output power. In summary, the 3D monolithic carbon material coated with nano-TiO2 is a promising anode material.A 3D porous carbon monolithic electrode was prepared from a wax gourd by freeze-drying and carbonation and then coated with nano-TiO


Diarrhea has been a global health problem for centuries, and its treatment has become increasingly difficult due to the antibiotics overuse and resistance. Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs, however, the mechanism of quercetin alleviating LPS-induced duodenal inflammation remains elusive. The results demonstrated quercetin enhanced the inflammatory cell infiltration in the Peyer’s patch of the intestinal mucosa after LPS induction. The LPS-induced expressions of these inflammation-related factors were completely blocked by quercetin. Quercetin also decreased the protein expression of inflammatory factors after LPS induction. Quercetin could down-regulate autophagy gene expression, and decreased the protein expression after LPS induction. Quercetin treatment prevented LPS-induced increases in the gene expressions of programmed cell death factors; meanwhile, quercetin decreased the protein expression of caspase 1 and caspase 3 after LPS challenge. LPS reduced the gene expression of mucin 2, but upregulated the mRNA and protein expression of claudin 1, occludin, and ZO-1, which was balanced by quercetin. These evidences suggest quercetin can alleviate duodenal inflammation induced by LPS through modulating autophagy, programmed cell death, and intestinal barrier function.n = 120) were allocated to groups including control, PBS with or without alcohol, LPS (125 ng/egg) with or without quercetin , and quercetin groups . Fifteen day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the duodena of the embryos were collected for histopathological examination, RNA extraction and real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting. The results demonstrated quercetin enhanced the inflammatory cell infiltration in the Peyer’s patch of the intestinal mucosa after LPS induction. The LPS-induced expressions of these inflammation-related factors  were completely blocked by quercetin. Quercetin also decreased the protein expression of TLR4, IL-1β, MMP3, and MMP9 after LPS induction. Quercetin could down-regulate autophagy gene expression , and decreased the protein expression of ATG5, and LC3-1/LC3-2 after LPS induction. Quercetin treatment prevented LPS-induced increases of the gene expressions of programmed cell death factors ; meanwhile, quercetin decreased the protein expression of CASP1 and CASP3 after LPS challenge. LPS reduced the gene expression of mucin 2, but upregulated the mRNA and protein expression of claudin 1, occludin, and ZO-1, and this was balanced by quercetin. This evidence suggests that quercetin can alleviate duodenal inflammation induced by LPS through modulating autophagy, programmed cell death, intestinal barrier function.Diarrhea has been a global health problem for centuries, and the treatment has become increasingly difficult duo to the antibiotics overuse and resistance. Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs, however, the mechanism of quercetin alleviating LPS-induced duodenal inflammation remains elusive. Specific pathogen-free chicken embryos ( Escherichia coli, Shigella, Salmonella [Campylobacter, and Vibrio species among the most common pathogens. Salmonella pullorum is the major pathogen of chicken diarrhea that is harmful to the poultry industry in developing countries. Enterotoxigenic Escherichia coli is a major cause of post-weaning diarrhea in pigs and causes significant damage to the swine industry worldwide. The treatment caused by Gram-negative bacteria has become increasingly difficult due to the antibiotics overuse and resistance [Salmonella enterica typhimurium is one of the most frequent causal pathogen of food contamination through meat animals associated to diarrhea, representing a major disease burden worldwide. The outer membrane of Gram-negative bacteria comprises lipopolysaccharide (LPS), which induces inflammation initiating macrophage activation, complement induction, and tissue factor activation. LPS could induce nucleotide oligomerization domain-like receptor pyrin domain-containing protein3 gene expression in the small intestine and large intestine of chickens [Diarrhea has been a global health problem for centuries. Since 1990, diarrhea has been ranked among the top ten causes of morbidity and mortality in all age groups [lmonella , Campylosistance . Salmonechickens . The infchickens . CytokinRhizoma coptidis), danshen  and their active ingredients have been shown to possess antibacterial activity in vitro. Therefore, use of alternative medicines such as phytochemicals or TCM to treat diarrhea is feasible and would reduce antibiotic use in humans, livestock, and poultry industries. Antibiotics’ overuse and overprescription in human and veterinary medicine are common issues worldwide in the treatment and prophylaxis of bacterial infections. Residues of antibiotics in animal products may cause antibiotic resistance and allergies. Therefore, the reduction of antibiotics use in humans and livestock and poultry industries is urgently needed. In this context, it is imperative to search alternative medicines to meet this objective. There are many Chinese herbal medicines that could treat diarrhea. Traditional Chinese herbal medicines, such as Coptis Root  was created to promote and facilitate research of traditional Chinese medicines and drug discovery. TCMSP, together with other online databases, such as SwissTargetPrediction, OMIM, and GeneCards, as well as the Database for Annotation, Visualization and Integrated Discovery (DAVID), are powerful tools for predicting and analyzing drug targets and signaling pathways. https://omim.org/ (26 January 2021) and GeneCards (https://www.genecards.org/ (accessed on 6 February 2021). Genes predicted from the above databases were analyzed using the Cytoscape software(version 3.7.2), and gene targets of the ingredients matching the genes associated with diarrhea were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in the Database for Annotation, Visualization and Integrated Discovery (DAVID) to predict molecular biology functions, biology process, and cytology component, and signaling pathways. In this study, we selected Chinese herbal medicines with antidiarrheal effects from the TCM literature, identified active ingredients of the herbal medicines using the TCMSP database, then, gene targets of the active ingredients were predicted on SwissTargetPrediction, and genes associated with diarrhea were obtained on OMIM  database using the following screening criteria: (1) the oral bioavailability should be 30% or higher, and (2) drug-likeness should be greater than or equal to 0.18. Each selected Chinese herbal medicine contains 2 to 92 bioactive ingredients, yielding a total of 291 bioactive ingredients from the 28 Chinese herbal medicines. The 291 active ingredients were analyzed on Cytoscape (version 3.7.2). Three active ingredients, quercetin, kaempferol, and beta-sitosterol, were selected based on the highest networking node degrees. The selected Chinese herbal medicines were analyzed for bioactive ingredients in the Traditional Chinese Medicine Systems Pharmacology ). Potential genes associated with diarrhea, dysentery or bacterial dysentery were obtained from OMIM (https://omim.org/ (accessed on 26 Januray 2021) and GeneCard (https://www.genecards.org/ (accessed on 6 February 2021). The potential pharmacological targets of the 3 ingredients and the genes associated with diarrhea were analyzed using the Cytoscape software (3.7.2) for common genes from the 3 databases. Potential target genes of the identified ingredients, quercetin, kaempferol and beta-sitosterol, were predicted on SwissTargetPrediction (https://david-d.ncifcrf.gov/tools.jsp (accessed on 22 September 2020).The target genes were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in the Database for Annotation, Visualization and Integrated Discovery (DAVID) to predict molecular biology functions, biology processes, cytology components, and signaling pathways  was dissolved in phosphate-buffered solution (PBS) at 0.6 μg/mL (125 ng/egg). Quercetin  was dissolved in 100% ethanol at 50, 100 and 200 μM . LPS from Mycoplasma gallisepticum, Mycoplasma synoviae, Newcastle disease virus, reticuloendotheliosis virus, and Salmonella pullorum gallinarium. The fertilized eggs were individually weighed and divided into 10 groups, each group consisting of 4 replicates with 3 eggs per replicate. The eggs were incubated under standard conditions . All eggs were candled and weighed at embryonic day 7 and embryonic day 14 to eliminate undeveloped eggs. They were untreated or injected with 0.2 mL/egg of PBS, LPS , PBS and ethanol (0.2 mL each per egg), quercetin + LPS , and quercetin . Each treatment was administered to 15 day-old embryonated eggs by injection into the allantoic cavity according to the procedure described by a previous study [Because the chick genome demonstrates remarkable evolutionary conservation with mammals, the expression patterns of several genes and proteins are well-conserved between chick and mouse embryos. In addition, injection into the allantoic cavity of chicken embryos was an ideal method to avoid the interaction of environmental LPS and intestinal LPS from gut microorganisms. Therefore, the chicken embryos were selected for the present study. Specific pathogen-free babcock embryos (weight 56.76 ± 3.32 g) were provided by a chicken breeder . The antibodies to many viruses and bacteria were negative in these embryos, including adenovirus group I and group III, avian influenza type A, reovirus, anemia virus, fowl pox, infectious bronchitis virus, infectious bursal disease, infectious laryngotracheitis virus, lymphoid leucosis virus A, B, and J, Marek’s disease , At embryonic day 19, the duodenum of the embryos was collected for histological examination and RNA extraction was performed for real-time quantitative polymerase chain reaction (qPCR). The duodenum tissues for histological examination were processed by a routine method (see below). The sample for PCR was stored in liquid nitrogen until RNA extraction. The study was approved by the Jiangxi agricultural University Animal experiment Ethics Committee (JXAULL-2022002).Gut tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin blocks, sectioned to 3 μm-thick sections , mounted on slides, and stained with haematoxylin and eosin following established histology procedures. The slides were scanned by a Pannoramic DESK  with the panoramic scanner software.®One-step gDNA removal and cDNA Synthesis SuperMix kit  by a T100 thermal cycler  according to the manufacturer’s protocol. The mRNA levels of genes were determined by real-time quantitative PCR using a BioRad CFX384 Real-Time system  or BioRad CFX Connect Real-Time system . A total of 33 genes were selected to study the gene intestinal inflammatory factors, autophagy, apoptosis, necroptosis, and intestinal mucosal barrier function. The sequence of genes was obtained from the USA National Center for Biotechnology Information web , and the forward and reverse primers were obtained by Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 5 June 2021 to 15 September 2021). The primers are listed in ®Green qPCR SuperMix  and 0.2 µM of each primer (0.4 μL /primer). PCR reactions consist of 1 cycle at 94 °C for 30 s and 43 cycles at 94 °C for 5s and an annealing temperature of 60 °C for 15 s and 72 °C for 10 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The relative levels of target mRNA expression were calculated using the 2−∆∆Ct method.Total RNA was extracted from liquid nitrogen-frozen duodenum (50 mg) using the TransZol Up Plus RNA kit . Absorbance at 230, 260 and 280 nm was measured by spectrophotometry  for the assessment of RNA purity. The extract with both OD260/280 nm (2.07 ± 0.03) and OD260/230 nm ratios (2.20 ± 0.12) was acceptable for PCR analysis. First-strand cDNA was synthesized from total RNA (800 ng) with an EasyScriptImmunohistochemical investigations were carried out using an indirect method of peroxidase with a primary antibody specific for CASP1 , CASP3 , Claudin 1 , LC3-1/LC3-2 , MMP9 , and TLR4 .2O2 in room temperature for 25 min in dark place, then were washed in PBS (pH = 7.4) in a decolorizing shaker for 3 times (5 min per time) to eliminate endogenous peroxidase activity.Paraffin sections were deparaffinized and rehydrated, and sections were kept in epitope retrieval solution (pH = 6.0 citric acid), and heated and boiled with moderate power by a microwave oven for 8 min, cooled for 8 min, and heated with low power for 7 min, then were washed in PBS (pH = 7.4) in a decoloration shaker for 3 times (5 min per time). Serial sections were incubated with 3% HThey were then rinsed and blocked by 3% normal bovine serum albumin  in room temperature for 30 min. Sections were rinsed and incubated with a primary antibodies, anti-CASP1 (1:1000), anti-CASP3 (1:500), anti-Claudin 1 (1:500), anti-LC3-1/LC3-2 (1:200), anti-MMP9 (1:500), and anti- TLR4 (1:1000), in a moist chamber at 4 °C for 12 h. Sections were washed in PBS (pH = 7.4) in a decoloration shaker for 3 times (5 min per time) and centrifuged. Following this, the sections were rinsed and incubated for 50 min at room temperature in goat and anti-rabbit immunoglobulins (IgG) that were horseradish peroxidase (HRP)-conjugated . Sections were washed in PBS (pH = 7.4) in a decolorizing shaker for 3 times (5 min per time). The slides were incubated with diaminobenzidine solution  and we terminated the stain by running water. Then, they were counterstained by hematoxylin solution for 3 min and rinsed by running water. After rinsing in PBS, slides were dehydrated, mounted, and examined under a microscope  equipped with a Nikon Digital imaging system .g at 4 °C for 10 min, and we collected the supernatant. The total protein concentration of the dissolved lysate was quantified using the bicinchoninic acid protein assay kit . Protein loading buffer was added  and heated for 15 min at 100 °C, then cooled to room temperature, and stored in a −20 °C refrigerator. A sample of 20 μg protein was electrophoresed using polyacrylamide gel (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane using a wet transfer electrophoresis apparatus . The blots were then blocked with 5% skimmed milk PBST for 30 min followed by overnight binding with specific antibodies at 4 °C, incubation with the secondary antibody for 30 min, and extensive washing. The specific antibodies were used to detect TLR4 , IL-1β , MMP9 , MMP3 , CASP1 , CASP3 , claudin 1 , occludin , β-actin , and the secondary IgG HRP-conjugated antibody . The expression levels of the target proteins were normalized to β-actin.Fresh tissues were homogenized, washed in phosphate-buffered saline (PBS) solution and lysed in RIPA lysis buffer  supplemented with protease inhibitor  (RIPA:phenyl methyl sulfonyl fluoride: protein phosphatase inhibitor = 100:1:1). The homogenates were lysed on ice for 30 min, and vibrated once per 5 min, centrifuged at 14,170× SPSS software . The data are presented as mean ± standard deviation, and were analyzed using the paired T test method. Differences at p < 0.01 were considered significant. All data were statistically analyzed using the A total of 291 bioactive ingredients were found for 28 Chinese herbal medicines by TCMSP. Using Cytoscape interaction network analysis, the top three ingredients with the highest node degrees were quercetin , kaempferol  and beta-sitosterol  , which whttp://www.swisstargetprediction.ch/ (accessed from 2 July 2020). There were 99, 98, and 15 potential targets predicted for quercetin, kaempferol and beta-sitosterol, respectively. We also obtained genes for diarrhea, dysentery or bacterial dysentery on OMIM (https://www.omim.org/ (accessed on 26 January 2021) and GeneCards (https://www.genecards.org/ (accessed on 6 February 2021). The number of genes for diarrhea, dysentery or bacterial dysentery was 797 by OMIM, and 3253 by GeneCards. The potential biological targets of the three ingredients and genes associated with diarrhea were analyzed by Cytoscape network analysis to screen for common genes from all three databases. A total of 15 mutual target genes with a network node degree greater than four were identified . The molecular functions involved RNA polymerase II transcription factor activity, enzyme binding, nitric-oxide synthase regulator activity, receptor signaling protein tyrosine kinase activity and protein binding of molecular function. The estrogen signaling pathway and tumor necrosis factor (TNF) signaling pathway were the main pathways .When the LPS dose was 125ng, there was bleeding in lamina propria of intestinal epithelial cells; there was inflammatory cell infiltration in the Peyer’s patch in LPS group. In quercetin group, intestinal epithelial cells had no inflammatory cells infiltration. .As expected, LPS induced pattern recognition receptor TLR4 and nuclear factor kappa B subunit 1(NFKB1) mRNA expression, as well as the mRNA expression of inflammatory cytokine interferon gamma (IFNγ) and the chemoattractants interleukin-8 (IL-8), interleukin-6 (IL-6), and interleukin-1β (IL-1β), MMP3, and MMP9. The LPS-induced expression of these inflammation-related factors was completely blocked by quercetin at all doses. Quercetin or LPS had no effects on myeloid differentiation primary response 88 (MYD88), an inflammatory cytokine, and quercetin did not alter LPS-induced down-regulation of interleukin-10 (IL-10), an anti-inflammatory cytokine . The immAutophagy activity was significantly increased by LPS, as indicated by markedly increased mRNA expression of autophagy related gene 5 (ATG5), LC3-1, LC3-2, and serine/threonine kinase 11 (LKB1) . The immConsistent with the inflammation of duodenum, the death receptors Fas cell surface death receptor (Fas) and ligand (TNFα), B cell CLL/lymphoma 2 (Bcl-2), as well as the caspases , cell death regulator dynamin 1 like (Drp1), mitochondrial fission protein, and receptor interacting serine/threonine kinase 1 (RIPK1), involved in apoptosis, pyroptosis and necroptosis, were upregulated by LPS. Quercetin treatment significantly prevented LPS-induced increases of these programmed cell death factors . The dowLPS reduced the expression of mucin 2, a major mucus component of the intestinal villus, but considerably upregulated the epithelial tight junction tight proteins, zonula occludens-1 (ZO-1), claudin 1 and occludin . QuercetStreptococcus mutans [Salmonella enterica serotype typhimurium, and Escherichia coli [Salmonella enterica serotype typhimurium to simulate the intestinal inflammation, and revealed quercetin attenuated the intestinal inflammation. Zhang et al. found quercetin derived from the methanol extract of hawthorn presented antibacterial activity against Staphylococcus aureus [Syzygium aromaticum) had an antibacterial effect on multidrug-resistant bacteria, including Klebsiella pneumoniae and most Staphylococcus aureus strains [Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs; it has antioxidant, anti-inflammatory, hepatoprotective, and anti-cancer effects . It is rs mutans , Salmonehia coli . In the s aureus . We foun strains . Our stuThe intestinal mucosal barrier function is the first line of defense against pathogens, nutritional deficiency, and stress. The elements of mucosal barrier function have four parts: epithelia, mucus layer, mucosal immune system, and microbiota. Mucosal barrier function depends largely on dynamic balance of epithelia cell proliferation, differentiation, migration, apoptosis, and shedding. The renewal process of intestinal epithelia lasts approximately one week. Chicken embryos start to ingest the amniotic fluid around 17 days of incubation, stimulating the rapid development of intestinal mucosa . In our Salmonella enterica serovar pullorum infection [Salmonella enterica serotype typhimurium infection [The mucosal immune system consists of various types of leukocytes and dendritic cells. The recruitment and activation of leukocytes and dendritic cells will promote intestinal inflammation. Pattern recognition receptors such as TLR and nucleotide-binding oligomerization domain proteins (NOD) play a pivotal role in mediating host immune responses. Cumulative evidence has indicated that LPS could activate the TLR4/NFKB signaling pathway. TLR4 and NFKB gene expression increased in the intestine of chicks after nfection  and Salmnfection . Our stunfection . IL-1β snfection . LPS indnfection , which infection . As expeSalmonella typhimurium revealed a massive inflammatory response: the IL-1β and IL-8 were overexpressed [In addition, IL-10 production, derived from macrophages, dendritic cells, and T cells, increased after LPS challenge. IL-10 knock-out mice spontaneously develop intestinal inflammation . Accordixpressed , similarxpressed . It is pMYD88 is a signal mediator between TLR4 and NFKB , but itsSalmonella typhimurium infection for mice [Extracellular matrix (ECM) is a complex macromolecule network which is composed of fibers, proteoglycans, glycoproteins and polysaccharides , and plafor mice . Ulceratfor mice ,32. A prfor mice  and in mfor mice . Our resAutophagy refers to the process involving the decomposition of intracellular components via lysosomes. The intestinal autophagy is a complex system and plays a pivotal role in maintaining the homeostasis and integrity of the intestinal epithelium ,36. AutoMultiple members of mechanistic target of rapamycin kinase (MTOR) pathways can regulate the initiation of autophagy, such as AMPK and MTOR complex 1 (MTORC1) . AMPK isLPS and/or inflammation can induce all three pathways of programmed cell death, apoptosis, pyroptosis and necroptosis. Thus, we also investigated the expression of genes representing these cell death pathways. LPS induced all three programmed cell death pathways, as shown by the upregulation of the pyroptosis-related genes CASP1 and CASP12, apoptosis-related genes Bcl-2, CASP3, and Fas, and necroptosis-related genes Drp1 and RIPK1. As for other LPS-induced changes, quercetin blocked the upregulation of all three programmed cell death pathways. Apoptosis, programmed cell death, is evolutionarily conserved across species, occurring regularly to eliminate excessive and potentially compromised cells to ensure cellular homeostasis. LPS could induce intestinal inflammation and apoptosis . There wNecroptosis, a caspase-independent and lytic programmed cell death displaying intermediate features between necrosis and apoptosis, is characterized by cell rupture and the release of cellular contents, and induces inflammation. The key actors are the receptor-interacting- serine/threonine protein-kinases 1 (RIPK1) and dynamin 1 like (Drp1). RIPK1 not only is an upstream kinase for Drp1 phosphorylation , but alsThe intestinal mucosa provides a critical barrier of the body against unwanted foreign materials and its impairment may result in inflammation . The intQuercetin is a main constituent of Chinese herbal medicines commonly used in the treatment of diarrhea. We aimed to avoid the interaction of environmental LPS and intestinal LPS from gut microorganisms. Therefore, the chicken embryos were selected for the present study. We found that quercetin may inhibit intestinal inflammation, which is a major cause of diarrhea, through multiple molecular pathways, including by inhibiting inflammatory cytokines, receptors, and transcription factors involved in inflammation, modulating autophagy, maintaining mucosa barrier integrity, and down-regulating programmed cell death (graphic abstract). The effects of quercetin on other organs will be revealed in our other manuscripts. Meanwhile, it warrants further study to testify the effects of kaempferol and beta-sitosterol in terms of their antidiarrheal activity.


Lactococcus lactis could be utilized as a CpG-plasmid carrier (LACpG10) to enhance the host’s innate immunity against pathogenic invasion. In the present study, after obtaining LACpG10-HL from homogenized and lyophilized recombinant strain LACpG10, we treated primary chicken lymphocytes, two cell lines (HD11 and IPEC-J2), and chickens with LACpG10-HL, CpG plasmids (pNZ8148-CpG10), and other stimulants, and respectively confirmed the effects by conducting qRT-PCR, bacterial infection assays, and a zoological experiment. Our data showed that LACpG10-HL could induce excellent innate immunity by regulating autophagy reactions, cytokine expression, and motivating PRRs. Interestingly, despite having no direct antiseptic effect, LACpG10-HL improved the antibacterial capacities of lymphocytes and enterocytes at the first line of defense. Most importantly, water-supplied LACpG10-HL treatment reduced the average adverse event rates, demonstrating that LACpG10-HL maintained its excellent immunostimulatory and protective properties under farming conditions. Our research not only contributes to revealing the satisfactory effects of LACpG10-HL but also sheds new light on a cost-effective solution with optimal immune effects in green, antibiotic-free, and healthful husbandry farming.Antibiotics are broadly restricted in modern husbandry farming, necessitating the need for efficient and low-cost immunomodulatory preparations in antibiotic-free and healthful farming. As is known to all, CpG oligonucleotides  recognized by TLR9 in mammals (while TLR21 in avians) could collaborate with some united agent to induce stronger immune responses, but the cost is prohibitively expensive for farmers. Here, considering the coordination between TLR2 and TLR9/TLR21, we firstly proposed the idea that the well-fermented  Antibiotic abuse and misuse in human medicine have resulted in antimicrobial resistance (AMR), leading to the emergence of superbugs. Overuse of antibiotics on livestock contributes to the increased resistance of human pathogens exacerbating the public health crisis . In respStrikingly, synthetic oligonucleotides containing unmethylated CpG motifs (CpG-ODNs), which are recognized by Toll-like receptor (TLR) 9 in mammals while by TLR21 in avian and teleost, can precisely meet the demands ,4,5. AccLactic acid bacteria (LAB) are well-known probiotics for their competence in hindering the pathogens’ growth, maintaining the balance of intestinal microbiota and enhancing the availability of nutrients, and have been widely used in the food industry and medical applications ,25. Recoherpes simplex virus infection in the brain [Inspiringly, the proven synergistic connection between TLR2 and TLR9 suggests that LAB can be more than a carrier. Sørensen et al. reported that TLR2 and TLR9 functioning together can awaken the first line of host defense against he brain . Similarhe brain . Adequathe brain . Additiohe brain ,34. BaseL. lactis NZ9000 to attain the recombinant strain LACpG10. Later, after homogenization followed by lyophilization, the lyophilized powder of LACpG10 homogenate was obtained, which is referred to hereafter as “LACpG10-HL”. In vitro, the immunoenhancing effects and related mechanisms of LACpG10-HL were investigated respectively in chicken lymphocytes, IPEC-J2 and HD11. Ultimately, LACpG10-HL was fed to chickens orally to confirm the efficiency of LACpG10-LH under farming conditions. In summary, this study firstly proposed the idea that L. lactis could be utilized as a carrier and a cost-effective united agent of CpG-DNA to improve the therapeutic efficacies of CpG-DNA, and consequently provides a promising solution in the post-antibiotic era in the green, healthy, and sustainable development of husbandry.Initially, this study constructed recombinant plasmids containing CpG motifs, which were subsequently transformed into Sac I and Xba I. After confirming by sanger sequencing, we obtained the recombinant pNZ8148-CpG10 . Afterward, we homogenized the LACpG10 cells and then implemented lyophilization to attain the lyophilized powder of LACpG10 homogenates (LACpG10-HL) as our CpG agents for further analyses .Based on our previous studies, we have found that chicken lymphocytes were suitable for the preliminary evaluation of CpG-DNA . TherefoOur results showed that the cytokines and autophagy levels were both upregulated by the two CpG agents after a 30 h co-culture. Interestingly, compared with the plasmids alone, LACpG10-HL showed a higher expression level of IL-1β while less IFN-α, for the contained immune stimulus such as teichoic-acid and various proteins might change the immunoregulatory pattern . AdditioIn order to further clarify the antibacterial potentiality of LACpG10-HL, we detected the gene expressions of PRRs , includiPseudomonas aeruginosa, Shigella sonnei, Enterohemorrhagic Escherichia coli (EHEC), and Streptococcus pyogenes [In order to evaluate the effects of LACpG10-HL in infectious circumstances, pyogenes  were useP. aeruginosa, S. sonnei, EHEC, and S. pyogenes to detect whether they had direct antibacterial effects. As results are shown in Before being co-culture with cells, we evaluated the effects of each sample on the growth of Afterward, we examined the role of LACpG10-HL in defending pathogenic infections on intestinal cellularity. Firstly, we investigated the influence of LACpG10-HL treatment on the bacterial clearance capacity of the avian macrophage HD11. By viewing the HD11 bacterial clearance index , we founAdditionally, we investigated whether LACpG10-HL could effectively enhance the capacity of HD11 for phagocytosing pathogens. Therefore, we calculated the bacterial phagocytosis index in HD11 infected with pathogens at MOI = 10. Like the tendency of the HD11 bacterial clearance index, compared to the other groups in the HD11 bacterial phagocytosis index , LACpG10p < 0.01 vs. the other treatments). This observation revealed that LACpG10-HL better protected enterocytes from pathogens averting the direct damage to the intestine barrier, consistent with the HD11 results.Meanwhile, we also detected the effect of LACpG10-HL on the bacterial invasion of IPEC-J2. IPEC-J2, an intestinal enterocyte, is the initial barrier against pathogens in the intestine, which triggers us to uncover whether LACpG10-HL can better protect enterocytes from pathogens. Contrary to the former indices, the lower this IPEC-J2 bacterial invasion index, the stronger the ability of cells to resist external infection, as well as the enhancement of innate immunity . As the To further validate the observed phenomenon in this section, we determined the expression level of genes related to CpG-induced immunity  are excellent immunostimulants but whose production cost is prohibitively expensive for widespread animal farming use ,40,41. TP. aeruginosa, S. sonnei, EHEC, and S. pyogenes, and found that they had no direct inhibitory effect on the pathogens, implying that the antibacterial role of pNZ8148-CpG10 or LACpG10-HL is primarily dependent on the immunoregulatory capacities.To function in a similar role as antibiotics, we firstly assessed the direct anti-infectious capacity of the CpG plasmids or LACpG10-HL alone. We treated them to the four pathogens, including Therefore, the gene expressions of active molecules involved in innate immunity were elevated. Initially, we chose to look at the transcriptional responses of the related genes in primary chicken lymphocytes treated with the stimulants. Autophagy is a conserved catabolic process in all eukaryotic cell types related to lysosomal activity implicating in the direct elimination of microbes, control of inflammation, antigen presentation, lymphocyte homeostasis, and secretion of immune mediators ,43. MacrNeisseria gonorrhoeae and Streptococcus has also been reported [On the other hand, since bacterial infection is one of the classic instigators of inflammation , we tookreported ,51. Morereported ,53, and reported ,55. By treported ,56. To go a step further into the capacities of LACpG10-HL against infection and circumvent the defect that chicken lymphocytes are not ideal model cells for bacterial infection, we turned to the two cell lines (HD11 and IPEC-J2) with infections of four pathogenic bacteria to mimic the infectious conditions . Under tThe mechanism behind the stronger immunostimulatory efficacies of LACpG10-HL is comprehensible. Firstly, based on the direct proof of the homologous relationship and similar functional effects among TLR21 and TLR9 ,63, as wThus, the upstream sensors that trigger the expressions of the tested cytokines and autophagy proteins need to be detected. We tried to investigate the LACpG10-HL’s influences on PRRs. PRRs are a broad range of molecules, including viral sensors , LPS sensor (TLR4), LAB sensor (TLR2), and CpG sensor , that the organisms use to sense the invading pathogens, thereby initiate the anti-infectious immunity ,67. SpecL. lactic with CpG plasmids, produced from cost-effective fermentation [As is known to all, the daily farming environment is not aseptic nor avirulent. Therefore, to evaluate the efficacy of LACpG10-HL more exactly in animals, we conducted a zoological experiment on chickens under a normal farming scenario, where chickens may face different pathogens, including viruses. The much lower adverse event rates with LACpG10-HL treatment validated that the combination of NZ9000 components and pNZ8148-CpG10 had a stronger capacity in initiating the host innate immunity against pathogens under farming conditions, according to the cellular data including cytokines, autophagy proteins, and PRRs, plus the bacterial clearance, phagocytosis, and invasion indexes. At the same time, the better efficacy of the drinking water-supplied group suggested that utilizing LACpG10-HL delivered by drinking water could be a better method to reduce the immune functional loss in destructive conditions such as stomach acid, thus improving the effect of LACpG10-HL. Notably, the high cost of CpG-DNA has greatly restricted its broadly commercial utilization in farming, herein entation , has shoThe initial purpose of our study was to excavate the role of CpG plus LAB (LACpG10-HL) in green, antibiotic-free, and healthful husbandry. If it works, LACpG10-HL will provide an effective approach to help animals defend against invading pathogens without antibiotics utilization. At this point, we have reached our goal, but it should be noted that this study overall was to evaluate the healthful breeding serviceability of LACpG10-HL in vivo and in vitro. However, determining the molecular mechanisms underlying protective immunity elicited by LACpG10-HL has been challenging. Further investigation will be necessary to reveal how the LAB components boost the CpG-DNA efficacy and the interaction between the PRRs and involve distinct signal pathways triggered by LACpG10-HL, which we plan to go a further step into. Notwithstanding its limitation, in our research, we confirmed that LACpG10-HL indeed enhanced the antibacterial immunity and globally displayed clinical protections in organisms, developing a new solution in antibiotic-free and healthful husbandry farming.In conclusion, our study demonstrated that CpG plus LAB (LACpG10-HL) outperformed CpG-DNA alone in eliciting innate immunity, and LACpG10-HL showed satisfactory protections against pathogens in vivo and in vitro, showing that LAB could be utilized as an efficient CpG-united agent. The primary roles of LACpG10-HL were to motivate the initial sensors (PRRs) for pathogens and thus elicit corresponding immune responses, including cytokine expressions and antibacterial autophagy. Our demonstrations that LACpG10-HL had better immunostimulatory effects and opened the door to exploring a cost-effective solution with ideal immune outcomes in antibiotic-free and healthy husbandry farming.L. lactis strains and the preparation of the CpG agents and control stimulants; (2) the assessment of immunoenhancing effects of the stimulants in chicken lymphocytes and IPEC-J2; (3) detections of direct antibacterial activity of the stimulants on four pathogens; (4) determinations of indexes related to antibacterial immunity in IPEC-J2 and HD11; (5) healthful farming evaluations of the stimulants.As shown in ® Master qPCR Mix was acquired from Tsingke, Beijing, China. The enzymes and kits for biological operations were purchased from Takara  and Tsingke . Cell culture reagents were obtained from Gibco .SanPrep endotoxin-free plasmid extraction kit and SanPrep Column Plasmid Mini-Preps Kit were purchased from Sangon Biotech, Shanghai, China. Lymphocyte Separate Kit was purchased from Haoyang, Tianjin, China. RNAiso Plus was acquired from TaKaRa, Beijing, China. BeyoRT™ II First-Strand cDNA Synthesis Kit (RNase H minus) was purchased from Beyotime, Shanghai, China. 2 × TSINGKEXba I and Sac I restriction sites of pNZ8148 . Afterward, the plasmids were extracted from MC1061 with a SanPrep endotoxin-free plasmid extraction kit . Followed by electroporation, we constructed the recombinant strains LA8148 and LACpG10. Recombinant strains were cultivated in 200 mL of GM17 medium (M17 medium with 0.5% glucose) containing 10 µg/mL of chloramphenicol for 18 h. After that, bacteria were centrifugal resuspended in 10 mL of sterile water and homogenized at 4 °C and 1000 bar three times before freeze-dried. Additionally, according to Yao et al. protocol [The plasmid pNZ8148 was stored in our laboratory . Recombi pNZ8148 . The twoprotocol , we pretLactococcus lactis subsp. Cremoris NZ9000, Escherichia coli MC1061, Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa), Shigella sonnei CMCC 51592 (S. sonnei), Enterohemorrhagic Escherichia coli ATCC 35150 (EHEC) and Streptococcus pyogenes ATCC 19615 (S. pyogenes) [In this study, six kinds of bacteria, including yogenes) , were pryogenes) .6 cells/mL after isolation. Then, 1 mL of the cell suspension was added to each well of 24-well plates and cultured at 41 °C under 5% CO2. HD11 cells were maintained under the same conditions as the former. Moreover, IPEC-J2 cells were maintained in DMEM-F12 (1:1) with 10% FBS and 5% CO2 at 37 °C. Cell culture reagents were obtained from Gibco .Unless noted, cell culture conditions are the same as follows: NZ9000 and the recombinant strains were inoculated in GM17 medium (M17 medium with 0.5% glucose) at 30 °C, while the rest bacteria were cultivated in Luria–Bertani (LB) medium at 37 °C. The chicken lymphocytes were diluted in a complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U penicillin/mL and 100 μg streptomycin/mL) to a final concentration of 5.0 × 10In this section, three cytology experiments were performed to assess the immunoenhancing effects of the stimulants on cells under conditions without pathogens. Based on the perspective of in vitro cytology, the immunostimulatory functions of various plasmids and CpG agents were evaluated. Treatments for each experiment are as follows:Experiment 1: Initially, to determine the optimal stimulation time, we directly added three stimulants to the chicken lymphocytes. Grouping: (1) PBS, (2) pNZ8148-CpG10 (10 μg/mL), and (3) LACpG10-HL solution (containing 10 μg/mL pNZ8148-CpG10). Afterward, the cell culture plates of chicken lymphocytes were placed under suitable conditions for 6 h, 20 h, and 30 h. Ultimately, cells were collected by centrifugation at 2000 rpm for 3 min and preserved at −80 °C for later quantitative real-time PCR (qRT-PCR)  to detecExperiment 2: Additionally, two more control stimulants, including pNZ8148 and LA8148-HL, were added to confirm the immunostimulatory capacities. Grouping: (1) PBS, (2) pNZ8148 (10 μg/mL), (3) pNZ8148-CpG10 (10 μg/mL), (4) LA8148-HL solution (containing 10 μg/mL pNZ8148), and (5) LACpG10-HL solution (containing 10 μg/mL pNZ8148-CpG10). Except that the stimulation time was the best (30 h) determined by Experiment 1, the other experimental operations and detections were the same as in Experiment 1.6 cells/mL in a complete RPMI-1640 medium, and the cell suspension was added to a 6-well cell culture plate at 1 mL per well. The details of grouping were the same as described in Experiment 2. After treatments, IPEC-J2 cells were inoculated for 30 h, collected by centrifugation at 2000 rpm for 3 min, and preserved at −80 °C for later qRT-PCR  including RIG-Ⅰ and MDA5 , as well as TLR2, TLR4, TLR7, TLR8, and TLR9  . IPEC-J2 qRT-PCR . P. aeruginosa, S. sonnei, EHEC, and S. pyogenes  of pathogens after 24 h co-culture. Four pathogens, including pyogenes , were tr5 cells/well without antibiotics [8 CFU/mL in DMEM-F12 (1:1) . After the pretreatments, we conducted four infection experiments on the two cell lines to determine three indexes  and the relative mRNA expressions of IL-12, IFN-γ, IFN-α, and IL-1β. Detailed treatments for each experiment are described as follows:In this section, we focused on the antibacterial immunoactivities of the stimulants in HD11 and IPEC-J2 cells infected by pathogens. Before adding pathogens, HD11 and IPEC-J2 cells were seeded into 24-plates at 1 × 10ibiotics . When thExperiment 1: Evaluations of bacterial clearance in HD11.Pretreated HD11 cells were co-cultured with four pathogens at MOI  = 1 for 2 h. To calculate the bacterial clearance index, the infected HD11 cells were lysed with 0.5% deoxycholate. Then, the cells were washed and lysed in H₂O containing 0.1% Triton X-100. Ultimately, the bacteria were resuspended in PBS, and serial dilutions of this lysate were plated on LB agar for counting CFU. The bacterial clearance index was expressed in percentages using the following formulation: (CFU counts in stimulants-treated infected cells/CFU counts in PBS-treated infected cells) × 100 .Experiment 2: Evaluations of bacterial phagocytosis in HD11.Pretreated HD11 cells were co-cultured with four pathogens at MOI = 10 for 1 h. The infected IPEC-J2 cells were washed with PBS after harvesting and incubated for 30 min in media containing 20 ug/mL gentamicin  to kill extracellular bacteria. Then, the washed cells were lysed with 0.5% deoxycholate and lysis in H₂O containing 0.1% Triton X-100. The number of bacteria in lysates was determined by counting CFU on an LB agar plate. The bacterial phagocytosis index was expressed in percentages using the following formulation: (CFU counts in stimulants-treated infected cells/CFU counts in PBS-treated infected cells) × 100 .Experiment 3: Evaluations of bacterial invasion in IPEC-J2.Except that HD11 was replaced by IPEC-J2, and the other experimental operations were the same as described in Experiment 2 in Experiment 4: Detections of the relative mRNA expressions.Infected HD11 and IPEC-J2 cells in the previous experiments were also collected after corresponding co-cultures and washed three times with sterile PBS, by then stored at −80 °C to detect the relative mRNA expressions of IL-12, IFN-γ, IFN-α, and IL-1β with qRT-PCR .RNA extraction and cDNA synthesis were used to test the relative mRNA level of immune-related genes of the samples collected in ® Master qPCR Mix (Tsingke) following the manufacturer’s protocol using the primers as shown in −ΔΔCt method. The primers used in qRT-PCR were synthesized by Tsingke. All the samples were performed in triplicate, and results were recorded in mean ± standard deviation [qRT-PCR was performed on CFX96 real-time system  with 2 × TSINGKEeviation .Fast yellow chicken  was usedClinical signs and morbidity were examined daily for 16 days post immunization. The adverse event rates were determined by observations of each bird’s signs of drowsiness and huddling, runny nose, difficulty breathing, diarrhea, or any other morbidity ,77,78. All the experimental procedures were approved by the Institutional Animal Care and Use Committees (IACUC) of South China Agricultural University (SCAU), Guangzhou city, Guangdong province, China. No specific permits were required for this study described here. The study area is not privately owned or protected in any way, and the field of studies did not involve endangered or protected species.p-value < 0.05) and an extremely significant difference .All experiments were performed at least three repetitions, and the results are expressed as mean ± standard error of the mean (SEM). The data in this study are conducted by one-way analysis of variance (ANOVA) and Student’s t-test for independent samples in GraphPad Prism 8 software. All the data charts are drawn by GraphPad Prism 8 software. One and two symbols above the histogram represents a significant difference (