IL-6 Transcription Research Articles

Recombinant BoHV-5 glycoprotein (rgD5) elicits long-lasting protective immunity in cattle

BoHV-5 is a worldwide distributed pathogen usually associated with a lethal neurological disease in dairy and beef cattle resulting in important economic losses due to the cattle industry. Using recombinant gD5, we evaluated the long-duration humoral immunity of the recombinant vaccines in a cattle model. Here we report that two doses of intramuscular immunization, particularly with the rgD5ISA vaccine, induce long-lasting antibody responses. Recombinant gD5 antigen elicited tightly mRNA transcription of the Bcl6 and the chemokine receptor CXCR5 which mediate memory B cells and long-lived plasma cells in germinal centers. In addition, using an in-house indirect ELISA we observed higher and earlier responses of rgD5-specific IgG antibody and the upregulation of mRNA transcription of IL2, IL4, IL10, IL15, and IFN-γ in rgD5 vaccinated cattle, indicating a mixed immune response. We further show that rgD5 immunization protects against both BoHV -1 and -5. Our findings indicate that the rgD5-based vaccine represents an effective vaccine strategy to induce an efficient control of herpesviruses.

Network Pharmacology Study of Bioactive Components and Molecular Mechanisms of the Glycoside Fraction from Picrorhiza scrophulariiflora Against Experimental Colitis.

To explore the potential mechanism of glycosidic fraction of Picrorhiza scrophulariiflora Pennell (GPS) extract for the treatment of colitis using UPLC-QTOF-MS analysis, network pharmacology and experimental research. The active components of GPS extract were identified by UPLC-QTOF-MS analysis and extracted their targets from the databases, which was used for network pharmacology analysis. Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis was performed to discover potential therapeutic mechanisms, and the network pharmacology results were then validated by in vivo and in vitro experiments. The results showed that GPS extract significantly alleviated the clinical signs of colitis, including body weight, disease activity index, colon shortening, and colon tissue damage, and inhibited the transcription and production of colonic IL-1β and IL-6 in DSS-induced colitis mice. In vitro, GPS extract also significantly suppressed nitric oxide (NO) production, iNOS expression, IL-1β and IL-6 transcription of LPS-activated RAW 264.7 cells. Network pharmacology integrated with experimental validation identified that GPS extract significantly suppressed Akt, p38, ERK, and JNK phosphorylation in vivo and in vitro, and luteolin, apocynin, caffeic acid, caffeic acid methyl ester, luteoloside, picroside II, aucubin, cinnamic acid, vanillic acid, and sweroside were the main components responsible for the anti-inflammatory effect of GPS. These findings demonstrate that the potential anti-inflammatory effect of GPS extract against colitis is achieved through suppressing PI3K/Akt and MAPK pathways, and that the abovementioned active components mainly exerted its anti-inflammatory effect. The therapeutic effect of GPS extract on colitis is related to PI3K/Akt and MAPK pathways, which is a promising remedy for colitis therapy.

Prohibitin is Upregulated on Donor T cells in Murine Acute Graft-versus-Host Disease and Promotes Th17 Response

Abstract Introduction: Acute graft-versus-host disease (GVHD) is a T cell-mediated disorder and the main cause of non-relapse mortality in allogeneic hematopoietic cell transplantation recipients. Here, we investigate PHB in Th17-mediated inflammation and as a therapeutic target for acute GVHD. Methods: Irradiated mice received T cell depleted bone marrow cells and allogeneic or syngeneic splenocytes. At day 25 post-transplant, blood, spleen, liver, and colon were harvested and analyzed for surface PHB, IFNγ, and IL-17 on donor T cells by flow cytometry. Splenocytes were cultured in the presence of PHB pharmacological inhibitor rocaglamide (RocA) and analyzed for PHB and IL-17 expression in donor T cells. Naïve murine CD4 T cells were cultured in Th17 polarizing conditions with RocA. CRISPR/Cas9-mediated knockout of PHB was performed followed by evaluation of Il17a, Il17f, and Il22 gene expression by quantitative PCR. Results: Surface expression of PHB is increased in murine allogeneic donor T cells in liver, spleen, colon, and peripheral blood in acute GVHD. PHB-expressing splenic T cells secrete IL-17, which is reduced with treatment with a PHB inhibitor in ex vivo experiments. PHB inhibition of murine CD4 T cells in vitro impairs differentiation into the Th17 subtype resulting in reduced IL-17 secretion and downregulation of Il17a, Il17f, and Il22 genes. Knockout of PHB reduces transcription of Il17 and Il22 genes. Conclusions: These findings show relevancy of PHB in Th17-mediated inflammation and suggest a potential role for PHB in acute GVHD. To our knowledge, this study is the first to investigate PHB in acute GVHD, and our findings will offer greater support for clinical use of PHB inhibition to treat this fatal disease. Supported by grants from AAI (KS, PR), ACS (PR; RSG-22-053-01-IBCD), and NIH NCI (PR; R01 CA252469).

Dominant Negative Variants in IKZF2 Cause ICHAD Syndrome, a New Disorder Characterized by Immunodysregulation, Craniofacial Anomalies, Hearing Impairment, Athelia, and Developmental Delay

Helios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and the development and regulation of the immune system. Although predominantly recognized for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed at varying levels in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans. Here, we investigated two unrelated individuals with immune dysregulation combined with syndromic features including craniofacial dysmorphism, sensorineural hearing loss, and congenital abnormalities resulting from de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZF) of Helios. Proband 1 (p. Gly136_Ser191dup) had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios and Proband 2 (p.Gly153Arg) had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios. Functional studies confirmed that both these variant proteins are expressed, and that they interfere with the ability of the wild-type Helios protein to perform its canonical function—repressing IL2 transcription activity—in a dominant negative manner. Our study is the first to describe dominant-negative IKZF2 variants. These variants cause a novel genetic syndrome characterized by Immunodysregulation, Craniofacial anomalies, Hearing impairment, Athelia, and Developmental delay (ICHAD syndrome).

PD-1 limits differentiation and plasticity of Tc17 cells.

Blockade of surface co-inhibitory receptor programmed cell death-1 (PD-1; CD279) has been established as an important immunotherapeutic approach to treat malignancies. On a cellular level, PD-1 is demonstrated to be of particular importance in inhibiting differentiation and effector function of cytotoxic Tc1 cells (CTLs). Nevertheless, the role of PD-1 in modulating interleukin (IL)-17-producing CD8+ T-cells (Tc17 cells), which generally display suppressed cytotoxic nature, is not well understood. To evaluate the impact of PD-1 in Tc17 responses, we examined its functioning using different in vitro and in vivo models. Upon activation of CD8+ T-cells in Tc17 environment, we found that PD-1 was rapidly expressed on the surface of CD8+ T-cells and triggered a T-cell-internal mechanism that inhibited the expression of IL-17 and Tc17-supporting transcription factors pSTAT3 and RORγt. Expression of type17-polarising cytokine IL-21 and the receptor for IL-23 were also suppressed. Intriguingly, adoptively transferred, PD-1-/- Tc17 cells were highly efficient in rejection of established B16 melanoma in vivo and displayed Tc1 like characteristics ex vivo. When using IL-17A-eGFP reporter mice for in vitro fate tracking, IL-17A-eGFP expressing cells lacking PD-1 signaling upon re-stimulation with IL-12 quickly acquired Tc1 characteristics such as IFN-γ, and granzyme B expression, implicating lineage independent upregulation of CTL-characteristics that are needed for tumor control. In line with plasticity characteristics, absence of PD-1 signaling in Tc17 cells increased the expression of the stemness and persistence-associated molecules TCF1 and BCL6. Thus, PD-1 plays a central role in the specific suppression of Tc17 differentiation and its plasticity in relation to CTL-driven tumor rejection, which provides further explanation as to why the blockade of PD-1 is such an efficient therapeutic target for inducing tumor rejection.

C-Jun-mediated JMJD6 restoration enhances resistance of liver cancer to radiotherapy through the IL-4-activated ERK pathway.

Radiotherapy is widely used in the treatment of liver cancer, but the efficacy can be limited by radioresistance. In this study, we attempt to delineate the possible molecular mechanism of c-Jun-regulated Jumonji domain-containing protein 6/interleukin 4/extracellular signal-regulated kinase(JMJD6/IL-4/ERK) axis in radioresistance of liver cancer. The expression of c-Jun was quantified in liver cancer tissues and cell lines, and the results indicated that c-Jun was upregulated in liver cancer tissues and cells. We further illustrated the role of c-Jun following gain- and loss-of-function strategies in malignant phenotypes of liver cancer cells. It was established that c-Jun elevated JMJD6 expression and augmented the malignancy and aggressiveness of liver cancer cells. The in vivo effects of c-Jun on radioresistance in liver cancer were validated in nude mice, in response to IL-4 knockdown or the ERK pathway inhibitor, PD98059. In the presence of JMJD6 upregulation, the expression of IL-4 was elevated in mice with liver cancer, which enhanced the radiation resistance. Moreover, knockdown of IL-4 inactivated the ERK pathway, thereby reversing the radiation resistance caused by overexpressed JMJD6 in tumor-bearing mice. Taken together, c-Jun augments the radiation resistance in liver cancer by activating the ERK pathway through JMJD6-upregulated IL-4 transcription.

Paradoxical activation of chronic lymphocytic leukemia cells by ruxolitinib in vitro and in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by an aberrant cytokine network that can support tumor growth by triggering janus kinase (JAK)/STAT pathways. Targeting cytokine-signaling should then be a rational therapeutic strategy but the JAK inhibitor ruxolitinib failed to control and seemingly accelerated the disease in clinical trials. The effect of ruxolitinib on primary human CLL cells was studied in vitro and in vivo. Ruxolitinib increased phosphorylation of IRAK4, an important toll-like receptor (TLR)- signaling intermediate, in circulating CLL cells in vitro. It also enhanced p38 and NFKB1 phosphorylation while lowering STAT3 phosphorylation in CLL cells activated with TLR-7/8 agonists and IL-2. Among the cytokines made by activated CLL cells, high levels of IL-10 contributed strongly to STAT3 phosphorylation and inhibited TLR7 activity. Ruxolitinib limited TLR-mediated IL10 transcription and markedly reduced IL-10 production in vitro. It also decreased blood levels of IL-10 while increasing TNFα along with phospho-p38 expression and gene sets associated with TLR-activation in CLL cells in vivo. The bruton's tyrosine kinase inhibitor ibrutinib decreased IL-10 production in vitro but, in contrast to ruxolitinib, blocked initial IL10 transcription induced by TLR-signaling in vitro, decreased TNFα production, and deactivates CLL cells in vivo. These findings suggest the possible benefits of inhibiting growth factors with JAK inhibitors in CLL are outweighed by negative effects on potential tumor suppressors such as IL-10 that allow unrestrained activation of NFκB by drivers such as TLRs. Specific inhibition of growth-promoting cytokines with blocking antibodies or infusing suppressive cytokines like IL-10 might be better strategies to manipulate cytokines in CLL.

Competitive binding of CD226/TIGIT with poliovirus receptor regulates macrophage polarization and is involved in vascularized skin graft rejection

End-stage organ failure often requires solid organ transplantation. Nevertheless, transplant rejection remains an unresolved issue. The induction of donor-specific tolerance is the ultimate goal in transplantation research. Here, an allograft vascularized skin rejection model using BALB/c-C57/BL6 mice was established to evaluate the regulation of the poliovirus receptor signaling pathway via CD226 knockout (KO) or TIGIT-Fc recombinant protein treatment. In the TIGIT-Fc-treated and CD226KO groups, graft survival time was significantly prolonged, with a Treg cell proportion increase and M2-type macrophage polarization. Donor-reactive recipient T cells became hyporesponsive while responding normally after a third-party antigen challenge. In both groups, serum IL-1β, IL-6, IL-12p70, IL-17A, TNF-α, IFN-γ, and monocyte chemoattractant protein-1 levels decreased, and the IL-10 level increased. In vitro, M2 markers, such as Arg1 and IL-10, were markedly increased by TIGIT-Fc, whereas iNOS, IL-1β, IL-6, IL-12p70, TNF-α, and IFN-γ levels decreased. CD226-Fc had the opposite effect. TIGIT suppressed Th1 and Th17 differentiation by inhibiting macrophage SHP-1 phosphorylation and enhanced ERK1/2-MSK1 phosphorylation and nuclear translocation of CREB. In conclusion, CD226 and TIGIT competitively bind to PVR with activating and inhibitory functions, respectively. Mechanistically, TIGIT promotes IL-10 transcription from macrophages by activating the ERK1/2-MSK1-CREB pathway and enhancing M2-type polarization. CD226/TIGIT-PVR are crucial regulatory molecules of allograft rejection.

FIBRONECTIN TYPE III DOMAIN CONTAINING 5 MITIGATES CHONDROCYTE SENESCENCE, SUBCHONDRAL OSTEOCLAST FORMATION, AND OSTEOARTHRITIS DEVELOPMENT THROUGH INHIBITING INTERLEUKIN-6

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development.Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining.Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production.Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA.

Intracloacal Inoculation of Broiler Chickens with Clostridium perfringens Strains: Evaluation of Necrotic Enteritis Disease Development and Lymphoid Immune Responses.

Necrotic enteritis (NE) is an economically important disease of chickens. We have recently shown that inflammatory responses in chickens inoculated orally with virulent Clostridium perfringens were spatially regulated. Here, we used previously virulence-characterized netB+C. perfringens strains, avirulent CP5 and virulent CP18 and CP26, to assess the severity of NE and immune responses in broiler chickens when inoculated intracloacally. The results showed that CP18- and CP26-infected birds had a reduced weight gain and developed milder/less severe NE lesions, as determined by the gross lesions scores, suggesting a subclinical-grade infection. Gene expression analysis in infected birds revealed three statistically significant observations compared to uninfected-control: (1) Increased expression of anti-inflammatory/immunoregulatory interleukin (IL)-10/transforming growth factor (TGF)β in cecal tonsil (CT) and bursa of Fabricius in the CP18/CP26-infected groups. (2) Increased CT transcription of pro-inflammatory IL-1β, IL-6 and interferon (IFN)γ and decreased Harderian gland (HG) expression of IFNγ in the CP18/CP26-infected birds. (3) Increased HG or bursal expression of IL-4 and IL-13 in CP5-infected birds. Collectively, intracloacal C. perfringens inoculation seems to induce a highly regulated inflammatory response in the CT and other mucosal lymphoid organs and an intracloacal infection model may be useful in evaluating immune responses in chickens with subclinical NE.

In ovo HVT vaccination enhances cellular responses at hatch and addition of poly I:C offers minimal adjuvant effects

In ovo vaccination with herpesvirus of turkey (HVT) hastens immunocompetence in chickens and the recommended dose (RD) of 6080 plaque-forming-units (PFU) offers the most optimal effects. In previous studies conducted in egg-type chickens, in ovo vaccination with HVT enhanced lymphoproliferation, wing-web thickness with phytohemagglutinin-L (PHA-L), and increased spleen and lung interferon-gamma(IFN-γ) andToll-like receptor 3 (TLR3) transcripts. Here, we evaluated the cellular mechanisms by which HVT-RD can hasten immunocompetence in one-day-old meat-type chickens, and also determined if HVT adjuvantation with a TLR3 agonist, polyinosinic-polycytidylic acid (poly(I:C)), could enhance vaccine-induced responses and provide dose-sparing effects. Compared to sham-inoculated chickens, HVT-RD significantly increased transcription of splenic TLR3 and IFN γ receptor 2 (R2), and lung IFN γ R2, while the splenic IL-13 transcription was found decreased. Additionally, these birds showed increased wing-web thickness following PHA-L inoculation. The thickness was due to an innate inflammatory cell population, CD3+ T cells, and edema. In another experiment, HVT-1/2 (3040 PFU) supplemented with 50 μg poly(I:C) [HVT-1/2+poly(I:C)] was administered in ovo and immune responses were compared with those produced by HVT-RD, HVT-1/2, 50 μg poly(I:C), and sham-inoculated. Immunophenotyping of splenocytes showed HVT-RD induced a significantly higher frequency of CD4+, CD4+MHC-II+, CD8+CD44+, and CD4+CD28+ T cells compared to sham-inoculated chickens, and CD8+MHC-II+, CD4+CD8+, CD4+CD8+CD28+, and CD4+CD8+CD44+ T cells compared to all groups. Treatment groups, except HVT-1/2+poly(I:C), had significantly higher frequencies of γδ T cells and all groups induced significantly higher frequencies of activated monocytes/macrophages, compared to sham-inoculated chickens. Poly(I:C)-induced dose-sparing effect was only observed in the frequency of activated monocytes/macrophages. No differences in the humoral responses were observed. Collectively, HVT-RD downregulated IL-13 transcripts (Th2 immune response) and had strong immunopotentiation effects on innate immune responses and the activation of T cells. However addition of poly(I:C) offered a minimal adjuvant/dose-sparing effect.

Immunomodulation in the intestinal mucosa of mice supplemented with Lactobacillus rhamnosus (ATCC 7469) and infected with Toxocara canis

Toxocariasis is an anthropozoonosis caused by the helminth Toxocara canis that shows different clinical manifestations as visceral, ocular, or neurological toxocariasis forms. Probiotics have been studied as alternatives to prevent and treat this parasitosis. Lactobacillus rhamnosus is a prospect that presents immunomodulatory activity that acts to strengthen the intestinal barrier. In this context, the main objective of this study was to evaluate the protective capacity and immunomodulatory action of the probiotic Lactobacillus rhamnosus at the level of the intestinal mucosa in different stages of T. canis infection (acute and chronic). Mice were supplemented by oral gavage with 1×107 UFC/mL L. rhamnosus for 15 days before inoculation with 100 embryonated eggs of T. canis. Euthanasia of mice was conducted at three different time points: 2, 15 and 30 days post-inoculation (PI). The brain, lungs and liver were collected to evaluate the intensity of infection. The small intestines were removed, and mucosal cells of the duodenum were collected to perform gene analysis of IFN-γ, IL-10, IL-4 and IL-13 by real-time polymerase chain reaction (qPCR). Jejunum and ileum segments were analysed by histological techniques. A reduction of 51% in infection intensity was observed in the tissue of supplemented animals evaluated 2 days PI; however, analysis of groups 15 and 30 days PI did not show a protective effect. The intestinal mucosa of supplemented animals presented an inflammatory process that initiated at 2 days PI, persisted at 15 days PI and had regressed at 30 days PI. IL-13 transcription was increased in the probiotic group 2 days after supplementation ended; however, the same increase was not observed in the group that was supplemented and infected. Toxocara canis modulated the local immune system, with suppression of IFN-γ at 2 days PI and increased levels of IL-4 and IL-10 at 15 days PI. These results indicate that, under the studied conditions, the protective effect of Lactobacillus rhamnosus against infection caused by T. canis is not related to IL-4, IL-10 or IFN-γ but could be influenced by IL-13 action at 2 days PI. The probiotic stimulated immune cell recruitment to the intestinal mucosa, which can be involved in the diminished capacity of larval penetration in the mucosa, resulting in the reduced infection intensity observed during acute infection.

From helping to regulating – A transcriptomic profile of Ifng+ Il10+ Il21+ Cd4+ Th1 cells indicates their role in regulating inflammation during experimental trypanosomosis

IntroductionTrypanosoma evansi parasite infections cause a chronic animal wasting disease called Surra, and cases of atypical Human Trypanosomosis (aHT). In experimental models, T. evansi infections are hallmarked by the early onset of excessive inflammation. Therefore, balancing the production of inflammatory cytokines by anti-inflammatory IL-10 is crucial for prolonged survival.MethodsTo improve the understanding of trypanosomosis induced immunopathology, we used scRNA-seq data from an experimental chronic T. evansi infection mouse model, resembling natural infection in terms of disease characteristics. Results and discussionFor the first time, obtained results allowed to assess the transcriptomic profile and heterogeneity of splenic CD4+ T cell subsets, during a trypanosome infection. Here, the predominant subpopulation of T cells was represented by Tbx21(T-bet)+Ccr5+ Id2+ type 1 helper T cells (Th1), followed by Icos+ Cxcr5+Follicular T helper cells (Tfh) and very minor fraction of Il2ra(CD25)+Foxp3+ regulatory T cells (Tregs). Interestingly, the profile of Th1 cells shows that besides Ifng, these cells express high levels of Il10 and Il21, coding for anti-inflammatory and immunoregulatory cytokines. This coincides with the elevated expression of key genes involved in IL-10 and IL-21 secretion pathway such as Stat1 and Stat3, as well as the transcriptional factors Prdm1 (Blimp 1), and Maf (c-Maf). In contrast, there is virtually no IL-10 transcription detected in the Treg population. Finally, differential gene expression and gene ontology analysis of infection-induced Ifng+ Il10+ Il21+ Th1 cells highlights their suppressive function on T cell activation, differentiation and INF-γ production itself. This indicates that during trypanosome infections, the Ifng+ Il10+ Il21+ Th1 cells, rather than Tregs, assume an immune regulatory role that is needed for dampening inflammation.

MiR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis.

Canine leishmaniasis (CanL) is a severe public health threat. Infected animals mediate transmission of the Leishmania protozoan to humans via the sandfly's bite during a blood meal. CanL progression depends on the degree of suppression of the immune response, possibly associated with microRNAs (miR), which can modulate mRNA translation into proteins and (consequently) regulate cell function. Increased miR-148a in splenic leukocytes (SL) of dogs with CanL was observed in previous studies, and in silico analysis, identified possible pathways involved in immune response regulation that are affected by this miR. Therefore, we evaluated the involvement of miR-148a in the regulation of TNF-α, IL-6, IL-12, IL-1β, iNOS, MHCII, CD80, CD3, T-bet, and GATA-3 transcription factors and their relationship with parasite load in SL of dogs with CanL. Splenic leukocytes obtained from healthy and diseased dogs were transfected with miR-148a mimic and inhibitor oligonucleotides. After 48 hours, expression levels of MHCII, CD80, iNOS, CD3, T-bet, and GATA-3 were evaluated by flow cytometry, and concentrations of TNF-α, IL-12, IL-6, and IL-1β were measured in culture supernatants by capture enzyme-linked immunosorbent assays. Transfection of SL with miR-148a mimics decreased iNOS levels in cells and TNF-α, IL-6, and IL-12 in the supernatants of cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in SL cells. These results suggest a direct or not regulatory role of this miR in the immune response to Leishmania infantum infection. We conclude that miR-148a can modulate immune responses by regulating inflammatory cytokines during CanL. Our results contribute to understanding the complex host/parasite interaction in CanL and could assist the development of treatments.

Vimentin affects inflammation and neutrophil recruitment in airway epithelium during Streptococcus suis serotype 2 infection

Streptococcus suis serotype 2 (SS2) frequently colonizes the swine upper respiratory tract and can cause Streptococcal disease in swine with clinical manifestations of pneumonia, meningitis, and septicemia. Previously, we have shown that vimentin, a kind of intermediate filament protein, is involved in the penetration of SS2 through the tracheal epithelial barrier. The initiation of invasive disease is closely related to SS2-induced excessive local inflammation; however, the role of vimentin in airway epithelial inflammation remains unclear. Here, we show that vimentin deficient mice exhibit attenuated lung injury, diminished production of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and the IL-8 homolog, keratinocyte-derived chemokine (KC), and substantially reduced neutrophils in the lungs following intranasal infection with SS2. We also found that swine tracheal epithelial cells (STEC) without vimentin show decreased transcription of IL-6, TNF-α, and IL-8. SS2 infection caused reassembly of vimentin in STEC, and pharmacological disruption of vimentin filaments prevented the transcription of those proinflammatory cytokines. Furthermore, deficiency of vimentin failed to increase the transcription of nucleotide oligomerization domain protein 2 (NOD2), which is known to interact with vimentin, and the phosphorylation of NF-κB protein p65. This study provides insights into how vimentin promotes excessive airway inflammation, thereby exacerbating airway injury and SS2-induced systemic infection.

P080 Mucosal clock and inflammation regulatory genes are disrupted in treatment naïve pediatric patients with ulcerative colitis

Abstract Background The human circadian clock is present in cells throughout the body. It consists of CLOCK and BMAL1 that heterodimerize and bind to E-box sequences to mediate transcription of a large number of genes, including Periods (PERs) and Cryptochromes (CRYs). PERs and CRYs constitute part of the negative feedback loop and inhibit CLOCK:BMAL1-mediated transcription. Recently, we have shown that patients with active ulcerative colitis (UC) display tissue-specific misalignment of the molecular clock that reverts to normal following effective treatment. Several circadian clock genes also function as anti-inflammatory regulators. RORa, PPARa and PPARg are positive regulators of the clock mechanism and induce the expression of IkB, a suppressor of NFkB. In the same manner, PCG1a induces transcription of IL10. Our aim was to characterize the expression of these regulators compared to that of core clock genes and determine their correlation in UC patients. Methods Clock gene expression patterns using whole transcriptome RNA sequencing were retrieved from the IBD Transcriptome and Metatranscriptome Meta-Analysis platform (TaMMA IBD). We compared rectal biopsy gene expression of 12 clock genes and their isoforms (AMPK, BMAL1, CLOCK, CRY1, CRY2, PER1, PER2, PCG1a, PPARa, PPARg, REV-ERB, RORa, RORg and SIRT1) in 206 treatment naïve pediatric patients with UC (age 12.9±3.2; 54% male) and 20 healthy controls (age 13.9±3.3; 45% male). Spearman correlations were calculated between all 12 clock genes within each of the UC and control cohorts. Results Core clock genes: BMAL (p<0.0001), CLOCK (p<0.01) and CRY1 (p<0.0001), and the anti-inflammatory regulator gene RORa (p<0.0001) were upregulated in UC patients compared to controls. In contrast, expression of other anti-inflammatory regulatory genes: PCG1a (p<0.0001), PPARa (p<0.01), PPARg (p<0.0001) and RORg (p<0.0001) was decreased. Clock gene expression levels of healthy controls show discordant correlations compared to UC patients. For example, correlations between BMAL/REV-ERBa as well as PPARg/REV-ERBa were inverted between controls and UC patients (r=-0.44 vs. r=0.13 and r =-0.4 vs. r=0.3, respectively). Figure 1. Expression of clock and inflammation regulatory genes in UC patients vs. healthy controls. Conclusion Core clock gene expression was disrupted, and anti-inflammatory regulator gene expression was decreased in patients with active UC vs. controls. The demonstration of discordant correlations of some positive vs. negative feedback loop genes in UC highlights the clock disruption in active inflammation. Since these clock regulators also ameliorate inflammation, their reduced expression may explain not only clock dysregulation but also increased tissue inflammation.

Mucosal and systemic lymphoid immune responses against Clostridium perfringens strains with variable virulence in the production of necrotic enteritis in broiler chickens

ABSTRACT Necrotic enteritis (NE), caused by Clostridium perfringens, is an economically important disease of chickens. Although NE pathogenesis is moderately well studied, the host immune responses against C. perfringens are poorly understood. The present study used an experimental NE model to characterize lymphoid immune responses in the caecal tonsils (CT), bursa of Fabricius, Harderian gland (HG) and spleen tissues of broiler chickens infected with four netB+ C. perfringens strains (CP1, CP5, CP18, and CP26), of which CP18 and CP26 strains also carried the tpeL gene. The gross and histopathological lesions in chickens revealed CP5 to be avirulent, while CP1, CP18, and CP26 strains were virulent with CP26 being “very virulent”. Gene expression analysis showed that, while the virulent strains induced a significantly upregulated expression of pro-inflammatory IL-1β gene in CT, the CP26-infected birds had significantly higher CT transcription of IFNγ and IL-6 pro-inflammatory genes compared to CP5-infected or uninfected chickens. Furthermore, CP26 infection also led to significantly increased bursal and HG expression of the anti-inflammatory/regulatory genes, IL-10 or TGFβ, compared to control, CP5 and CP1 groups. Additionally, the splenic pro- and anti-inflammatory transcriptional changes were observed only in the CP26-infected chickens. An antibody-mediated response, as characterized by increased IL-4 and/or IL-13 transcription and elevated IgM levels in birds infected with virulent strains, particularly in the CP26-infected group compared to uninfected controls, was also evident. Collectively, our findings suggest that lymphoid immune responses during NE in chickens are spatially regulated such that the inflammatory responses against C. perfringens depend on the virulence of the strain.

Lactobacilli Probiotics Modulate Antibacterial Response Gene Transcription of Dendritic Cells Challenged with LPS.

Probiotics are beneficial bacteria that may modulate the immune response by altering the maturation and function of antigen-presenting cells, such as dendritic cells. This study aimed to evaluate the antibacterial gene expression of dendritic cells challenged with LPS and probiotics. Immature dendritic cells were obtained from human CD14+ monocytes and challenged with E. coli LPS and probiotics Lacticaseibacillus rhamnosus (LR-32) and Lactobacillus acidophilus (LA-5) at a ratio DC:bacteria of 1:10. The analysis of gene expression was performed by RT-qPCR using the Kit RT2 human antibacterial response. In the supernatant, the cytokines secretion was determined by ELISA. Tukey post-ANOVA with p at 5% was used for statistical analysis. LPS showed the higher upregulation of 29 genes compared with the groups where probiotics were added to LPS, including genes related to an inflammatory response like BIRC3, CASP1, CCL5, CXCL1, IL12B, IL18, MYD88, NLRP3, RIPK1, and TIRAP. Similarly, LPS increased the transcription of genes enrolled with apoptosis such as CARD6, CASP1, IRF5, MAP2K1, MAP2K4, MAPK1, MYD88, NLRP3, RIPK2, TNF, TNFRSF1A, and XIAP when compared to probiotics groups (p < 0.05). Although probiotics decrease several genes upregulated by LPS, the transcription of encoded cytokines IL12A, IL12B, IL1B, IL6, CXCL8, and TNF genes was maintained upregulated by probiotics, except for IL18, which was downregulated by LA-5. LA-5 led to a higher transcription of IL1B, IL6, and CXCL-8 which was followed by the secretion of these proteins by ELISA. The results suggest that probiotics attenuate the transcription of inflammatory and immune response genes caused by LPS.

Patrinoside and Patrinoside A from Patrinia scabiosaefolia Improve Insulin Resistance by Inhibiting NF-κB, MAPK Pathways and Oxidative Stress in RAW264.7 and 3 T3-L1 Cells.

Patrinia scabiosaefolia, as traditional food and medicine plant, was used to treat appendicitis, enteritis, and hepatitis for thousand years in China. Patrinoside and patrinoside A isolated from P. scabiosaefolia could significantly improve insulin resistance (IR) by activating PI-3 K/AKT signaling pathway in our previous study. Since IR is closely related to inflammation, their anti-inflammatory activities in RAW264.7 inflammatory model induced by LPS and in 3 T3-L1 IR inflammatory model induced by TNF-α were evaluated to identify whether the effects on improving IR related to anti-inflammatory activity. In RAW264.7 cells, patrinoside and patrinoside A significantly inhibited the transcription and secretion of inflammatory mediators NO, TNF-α, and IL-6. Western blot analysis showed that the significant inhibition of phosphorylation of IκB and P65 and P38, ERK and JNK suggested that the effects were exerted through NF-κB pathway and MAPK pathway. In 3 T3-L1 cells, patrinoside and patrinoside A also inhibited the activation of NF-κB and MAPK pathways through inhibiting the transcriptions of inflammatory cytokines IL-6 and chemokines MCP-1 and MIP-1α. These events resulted in the inhibition of macrophages migration to adipocytes. In addition, patrinoside and patrinoside A ameliorated oxidative stress by inhibiting ROS release in LPS-stimulated RAW264.7 cells. In conclusion, patrinoside and patrinoside A could active PI-3 K/AKT pathway, inhibit NF-κB pathway, MAPK pathway, and improve oxidative stress, which showed multipathways on improving IR. These results provided the scientific basis for material basis and mechanism on improving IR of P. scabiosaefolia.

Fibrinogen primes the microglial NLRP3 inflammasome and propagates pro-inflammatory signaling via extracellular vesicles: Implications for blood-brain barrier dysfunction

The brain's response to acute injury is characterized by increased permeability of the blood-brain barrier (BBB) and pro-inflammatory microglia signaling, both of which have been linked to poor cognitive outcomes and neurological disease. The damaged BBB has increased leakiness, allowing serum proteins like fibrinogen into the brain, which interacts with local cells in a deleterious manner. At the same time, in response to injury, microglia demonstrate increased NLRP3 inflammasome activity and heightened release of pro-inflammatory cytokines. The relationship between increased fibrinogen uptake and microglial inflammasome signaling in the injured brain has not been well described. In this work, we investigate fibrinogen mediated NLRP3 inflammasome priming of BV-2 cells and primary adult microglia and propose a role for extracellular vesicles (EVs) as propagators of this interaction. Following exposure to fibrinogen microglia significantly upregulate transcription of IL-1β, IL-6, NLRP3 and other pro-inflammatory cytokines which was sustained by repeated fibrinogen exposure. Inhibition of fibrinogen mediated NLRP3 signaling was achieved at the transcriptional and assembly level using cannabidiol (CBD) and the NLRP3 inhibitor MCC950, respectively. EVs released following NLRP3 priming carry IL-1β, IL-18 mRNA and fibrinogen, propagate inflammatory signaling and can be detected in the circulation following BBB disruption in a preclinical stroke model. In conclusion, the interplay between fibrinogen extravasation, microglial NLRP3 signaling, and EV release can perpetuate chronic pro-inflammatory signaling and represents a novel method of inflammatory propagation.