IFN-γ Transcript Levels Research Articles

Sepsis Impairs IFN-γ Production in CD8 T Cells through Changes in Local Chromatin Landscape.

Sepsis is a complex condition of inflammatory and immune dysregulation, triggered by severe infection. In survivors, chronic inflammation and immune dysregulation linger, facilitating the emergence of infections. CD8 dysfunction contributes to immunosuppression in sepsis survivors. We devised an animal model that enabled us to identify and analyze CD8-intrinsic defects induced by sepsis. We adoptively transferred CD45.1 CD8 OT-I T cells into CD45.2 congenic mice and subjected them to cecal ligature and puncture, to induce abdominal sepsis. One month later, we isolated the transferred CD8 cells. Surface marker expression confirmed they had not been activated through the TCR. CD8 OT-I T cells isolated from septic (or sham-operated) mice were transferred to second recipients, which were challenged with OVA-expressing Listeria monocytogenes. We compared effector capacities between OT-I cells exposed to sepsis and control cells. Naive mice that received OT-I cells exposed to sepsis had higher bacterial burden and a shorter survival when challenged with OVA-expressing L. monocytogenes. OT-I cells isolated from septic mice produced less IFN-γ but had conserved activation, expansion potential, and cytotoxic function. We observed lower transcript levels of IFN-γ and of the long noncoding RNA Ifng-as1, a local regulator of the epigenetic landscape, in cells exposed to sepsis. Accordingly, local abundance of a histone modification characteristic of active promoter regions was reduced in sepsis-exposed CD8 T cells. Our results identify a mechanism through which inflammation in the context of sepsis affects CD8 T cell function intrinsically.

Cu(NN1)2]ClO4, a Copper (I) Complex as an Antimicrobial Agent for the Treatment of Piscirickettsiosis in Atlantic Salmon.

Piscirickettsia salmonis is the pathogen that most affects the salmon industry in Chile. Large quantities of antibiotics have been used to control it. In search of alternatives, we have developed [Cu(NN1)2]ClO4 where NN1 = 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one. The antibacterial capacity of [Cu(NN1)2]ClO4 was determined. Subsequently, the effect of the administration of [Cu(NN1)2]ClO4 on the growth of S. salar, modulation of the immune system and the intestinal microbiota was studied. Finally, the ability to protect against a challenge with P. salmonis was evaluated. The results obtained showed that the compound has an MIC between 15 and 33.9 μg/mL in four isolates. On the other hand, the compound did not affect the growth of the fish; however, an increase in the transcript levels of IFN-γ, IL-12, IL-1β, CD4, lysozyme and perforin was observed in fish treated with 40 μg/g of fish. Furthermore, modulation of the intestinal microbiota was observed, increasing the genera of beneficial bacteria such as Lactobacillus and Bacillus as well as potential pathogens such as Vibrio and Piscirickettsia. Finally, the treatment increased survival in fish challenged with P. salmonis by more than 60%. These results demonstrate that the compound is capable of protecting fish against P. salmonis, probably by modulating the immune system and the composition of the intestinal microbiota.

Abstract 1404: Blood-based transcriptomics provides novel insights into Sjögren's syndrome-associated lymphomagenesis

Abstract Sjögren's syndrome (SS) is a debilitating autoimmune disorder that carries the highest risk of progressing to lymphoma among all autoimmune diseases, with up to 10% of patients developing SS-associated lymphoma. However, the underlying mechanisms are unknown, and there are currently no actionable targets for prevention and therapeutic intervention.To elucidate the mechanisms associated with SS-lymphoma, we performed RNA-Seq in peripheral blood from SS patients without (n=11) or with lymphoma (n=8). Differential gene expression and pathway analysis identified TNF-α signaling via NF-κB, inflammatory response and interferon gamma (IFN-γ) response as the top pathways enriched in SS-lymphoma patients. Interestingly, the enriched IFN-γ response pathway is consistent with our published work identifying elevated IFN-γ transcript levels in salivary gland tissues from SS patients with lymphoma. Central to SS pathogenesis is the elevation of TNF-α signaling, triggering NF-κB pathway activation and subsequent inflammation, along with inflammasome activation, in line with prior observations. Our data reveal significant upregulation of these pathways in SS-lymphoma patients, which together with the inflammatory response and IFN-γ pathway enrichment, support the idea that persistent autoimmune-related chronic inflammation fosters a microenvironment conducive to lymphomagenesis.Next, we sought to assess the impact of SS immune landscape in SS-lymphoma by performing an immune modular analysis using an established framework of 97 whole blood transcriptomics modules (Pascual), derived from transcripts co-expressed in blood across various autoimmune conditions. This analysis revealed a significant downregulation of T-cell and enrichment of B-cell modules in SS-lymphoma compared to SS patients. These findings are consistent with our biopsy results, showing lower T cell counts in high-grade vs. low-grade SS patients. As patients with high-grade SS are more likely to develop SS-lymphoma, these data possibly suggest that suppressed T-cell responses are involved in lymphomagenesis. In addition, the significant enrichment in B-cell transcriptional modules mirrors the observed clinical increase in B cell counts during the transition to SS-lymphoma. Germline mutations in B-cells are thought to drive the clonal expansion of malignant B cells, which may contribute to the observed increase of B cell transcriptomes in SS patients developing lymphoma. We are currently investigating the gene mutation landscape in these datasets to better understand their contribution to SS- lymphomagenesis.This study provides preliminary evidence that chronic inflammation along with dysregulation of T and B compartments in SS significantly contributes to lymphoma progression, emphasizing the need for further research to uncover the molecular basis and develop early diagnostic/intervention tools. Citation Format: Ada Gjyrezi, Charalampos Skarlis, Paul Zumbo, Doron Betel, Ioanna E. Stergiou, Michael Voulgarelis, Clio P. Mavragani, Paraskevi Giannakakou. Blood-based transcriptomics provides novel insights into Sjögren's syndrome-associated lymphomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1404.

Oral administration of a new copper (I) complex with coumarin as ligand: modulation of the immune response and the composition of the intestinal microbiota in Onchorhynchus mykiss.

[Cu(NN1)2]ClO4 is a copper (I) complex, where NN1 is an imine ligand 6-((quinolin-2-ylmethylene) amino)-2H-chromen-2-one obtained by derivatization of natural compound coumarin, developed for the treatment of infectious diseases that affect salmonids. In previous research, we showed that the Cu(I) coordination complex possesses antibacterial activity against Flavobacterium psychrophilum, providing protection against this pathogen in rainbow trout during challenge assays (with an RPS of 50%). In the present study, the effects of administering [Cu(NN1)2]ClO4 to Oncorhynchus mykiss over a 60-days period were evaluated with regard to systemic immune response and its potential to alter intestinal microbiota composition. In O. mykiss, an immunostimulatory effect was evident at days 30 and 45 after administration, resulting in an increment of transcript levels of IFN-γ, IL-12, TNF-α, lysozyme and perforin. To determine whether these immunomodulatory effects correlated with changes in the intestinal microbiota, we analyzed the metagenome diversity by V4 16S rRNA sequencing. In O. mykiss, both [Cu(NN1)2]ClO4 and commercial antibiotic florfenicol had comparable effects at the phylum level, resulting in a predominance of proteobacteria and firmicutes. Nonetheless, at the genus level, florfenicol and [Cu(NN1)2]ClO4 complex exhibited distinct effects on the intestinal microbiota of O. mykiss. In conclusion, our findings demonstrate that [Cu(NN1)2]ClO4 is capable of stimulating the immune system at a systemic level, while inducing alterations in the composition of the intestinal microbiota in O. mykiss.

Leishmania infantum UBC1 in Metacyclic Promastigotes from Phlebotomus perniciosus, a Vaccine Candidate for Zoonotic Visceral Leishmaniasis.

Leishmania parasites cause outstanding levels of morbidity and mortality in many developing countries in tropical and subtropical regions. Numerous gene expression profiling studies have been performed comparing different Leishmania species’ life-cycles and stage forms in regard to their distinct infective ability. Based on expression patterns, homology to human orthologues, in silico HLA-binding predictions, and annotated functions, we were able to select several vaccine candidates which are currently under study. One of these candidates is the Leishmania infantum ubiquitin-conjugating enzyme E2 (LiUBC1), whose relative levels, subcellular location, in vitro infectivity in the U937 myeloid human cell model, and protection levels in Syrian hamsters against L. infantum infection were studied herein. LiUBC1 displays a low level of similarity with the mammalian orthologs and relevant structure differences, such as the C-terminal domain, which is absent in the human ortholog. LiUBC1 is present in highly infective promastigotes. Knock-in parasites overexpressing the enzyme increased their infectivity, according to in vitro experiments. Syrian hamsters immunized with the recombinant LiUBC1 protein did not show any parasite burden in the spleen, unlike the infection control group. The IFN-γ transcript levels in splenocytes were significantly higher in the LiUBC1 immunized group. Therefore, LiUBC1 induced partial protection against L. infantum in the Syrian hamster model.

Th17 Cells Contribute to the Pathology of Autoimmune Hypophysitis.

Autoimmune hypophysitis is classified as primary if its origin is idiopathic and secondary if it develops as a consequence of treatment with immune checkpoint inhibitors. Expanding use of immunotherapy has been paralleled by the increasing hypophysitis prevalence. However, understanding of the immune responses driving the disease remains limited. Using a mouse model of primary hypophysitis, we have identified CD4+ T lymphocytes to be the main pituitary-infiltrating immune cell population. Functional analysis showed that they display a Th17 and Th1/Th17 phenotype. To examine involvement of proinflammatory Th1, Th17, and Th1/17 subsets in hypophysitis, we have isolated RNA from the formalin-fixed paraffin-embedded pituitary specimens from 16 hypophysitis patients (three of whom had hypophysitis secondary to immune checkpoint inhibitors), 10 patients with adenoma, and 23 normal pituitaries obtained at autopsy. Transcript levels of IFN-γ, IL-17A, IL-4, IL-10, TGF-β, CD4, CD8α, and class II MHC transactivator were analyzed by the reverse transcription-quantitative PCR (RT-qPCR). Pituitary glands of patients with hypophysitis showed significantly higher IL-17A, CD4, and class II MHC transactivator mRNA levels compared with adenoma and normal pituitaries. All three secondary hypophysitis patients showed detectable IL-17A levels, but other cytokines were not detected in their pituitaries. Levels of IFN-γ, IL-4, IL-10, and TGF-β did not differ between the groups. TGF-β transcript was found in significantly fewer hypophysitis pituitaries (2 out of 16) compared with adenoma (7 out of 10) and normal pituitaries (11 out of 23). Presence of TGF-β in two hypophysitis patients was associated with significantly lower IL-17A mRNA levels compared with hypophysitis patients with no detectable TGF-β (p = 0.03).

Circulating microRNA Expression Profiling Identifies miR-125a-5p Promoting T Helper 1 Cells Response in the Pathogenesis of Hashimoto's Thyroiditis.

MicroRNAs (miRNAs) have emerged as key regulators of cellular processes by suppressing target mRNAs at the posttranscriptional level. However, little is known regarding the expression of miRNAs in peripheral blood mononuclear cells (PBMCs) from Hashimoto's thyroiditis (HT) patients. Therefore, 38 HT patients and 36 healthy volunteers were enrolled in this study to identify HT-mediated changes in miRNA expression. Over 1,000 dysregulated miRNAs and their biological functions in the HT patients were identified. Among them, miR-125a-5p expression was upregulated and inversely correlated with low levels of MAF, a transcription factor that inhibits Th1 cells activity and the production of IFN-γ. Luciferase assay results demonstrated that MAF is a direct target gene of miR-125a-5p. Moreover, the proportion of circulating Th1 cells and the transcript levels of IFN-γ were increased in the HT patients. MiR-125a-5p expression positively correlated with the proportion of circulating Th1 cells and the serum concentrations of anti-thyroperoxidase antibodies in the HT patients. Interestingly, knockdown of miR-125a-5p in CD4+ T cells resulted in an elevated level of MAF but decreased the proportion of Th1 cells and the transcript level of IFN-γ in vitro. Furthermore, upregulated miR-125a-5p and IFN-γ transcript levels and downregulated MAF expression were detected in thyroid tissues from HT patients. Receiver operating characteristic (ROC) curves suggested that miR-125a-5p has a crucial role in the HT. Our results demonstrate that the elevated levels of miR-125a-5p contribute to the Th1 cells response in the HT patients and may be involved in the pathogenesis of HT.

Erythromycin Prevents Elastin Peptide-Induced Emphysema and Modulates CD4+T Cell Responses in Mice.

PurposeElastin peptides (EP) can induce lung inflammation and emphysema. Erythromycin has been shown to decrease acute exacerbation frequency and delay lung function decline in chronic obstructive pulmonary disease patients and ameliorate emphysema in murine models; however, the mechanism remains unclear. We aimed to observe the preventive and immunomodulatory effects of erythromycin in a mouse model of EP-induced emphysema.MethodsIn the in vivo study, Balb/c mice were treated with EP intranasally on day 0, and then administered erythromycin (100 mg/kg) or vehicle orally on day 1, which was continued every other day. Mice exposed to cigarette smoke were used as an emphysema positive control. The severity of emphysema and inflammation in the lungs of EP-exposed mice with or without erythromycin treatment were observed on day 40 after EP administration. In the in vitro study, naïve CD4+T cells were isolated from healthy mice spleens and stimulated by EP with or without erythromycin incubation. Flow cytometry was used to measure the proportions of Th1, Th17, and Treg cells. ELISA was used to detect cytokine levels of IFN-γ, IL-17, IL-6, and TGF-β. Transcript levels of Ifnγ, IL17a, and Foxp3 were evaluated by qRT-PCR.ResultsAfter exposure to EP, Th1 and Th17 cell percentages and the levels of inflammatory cytokines increased in vivo and in vitro, while Treg cells decreased in vivo. Erythromycin reduced IFN-γ, IL-17, IL-6 inflammatory cytokines, MLI, and the inflammation score in the lungs of EP-exposed mice. In vitro, erythromycin also limited Th17 and Th1 cell differentiation and downregulated transcript levels of Ifnγ and IL17a in the EP-stimulated CD4+T cells.ConclusionThe Th1 and Th17 cell responses were increased in EP-induced emphysema. Prophylactic use of erythromycin effectively ameliorated emphysema and modulated CD4+T cells responses in EP-induced lung inflammation in mice.

Effects of dietary Lactobacillus rhamnosus JCM1136 and Lactococcus lactis subsp. lactis JCM5805 on the growth, intestinal microbiota, morphology, immune response and disease resistance of juvenile Nile tilapia, Oreochromis niloticus

The present study aimed to evaluate the individual and combined effects of Lactobacillus rhamnosus (LR) JCM1136 and Lactococcus lactis subsp. lactis (LL) JCM5805 on the growth, intestinal microbiota, intestinal morphology, immune response and disease resistance of juvenile Nile tilapia (Oreochromis niloticus). A total of 720 apparently healthy juvenile Nile tilapia (0.20 ± 0.05 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with JCM1136 (LR), JCM5805 (LL), and JCM1136 + JCM5805 (LR+LL) at 1 × 108 CFU/g basal diet for 6 weeks, followed by a basal diet for 1 week. After 6 weeks of feeding, the LL treatment significantly increased the growth and feed utilization of Nile tilapia when compared with the CK. Light microscopy and transmission electron microscopy images of the midgut revealed that probiotic supplementation significantly increased gut microvilli length and microvilli density compared to CK. The transcript levels of several key immune-related genes in the mid-intestine and liver of fish were analyzed by means of quantitative polymerase chain reaction (qPCR) at the end of the sixth week. The results showed the following: when compared to CK group, fish in LR had significantly increased transcript levels of IFN-γ, lyzc, hsp70 and IL-1β in the intestine; LL fish showed significantly increased expressions of TNF-α, IFN-γ, lyzc, hsp70 and IL-1β in the intestine and liver; and intestine lyzc, hsp70 and IL-1β and liver TNF-α, IFN-γ, hsp70 and IL-1β were significantly increased in LR+LL fish. Following a 6-week period of being fed probiotics or a control diet, the tilapia were challenged with an intraperitoneal injection of 20 μl of the pathogenic Streptococcus agalactiae (WC1535) (1 × 105 CFU/ml). The survival rates of the probiotic-fed groups were significantly higher than that of the CK group, and the LL group had the highest survival rate. High-throughput sequencing revealed a significantly higher presence of JCM5805 in the guts of LL fish during the period of probiotic application, but this was no longer detected in all LL samples 1 week post cessation of probiotic administration. Cessation of probiotic administration led to disorders of individual gut microbes within the LR and LL groups. Statistical analysis (LEfSe) demonstrated that three phyla, namely, Bacteroidetes, Fusobacteria and Actinobacteria were enriched in the CK group, while the abundance of Proteobacteria was greater in the probiotic-fed fish. At the genus level, Plesiomonas, which includes potential pathogens of fish, were significantly decreased in the probiotic-fed groups. In contrast, a significant increase of Rhizobium and Achromobacter, which can produce a variety of enzymes with cellulolytic and pectolytic activity, were observed in fish fed with probiotics, indicating that dietary probiotics were helpful in the propagation of some probiotic bacteria. Our data revealed that JCM1136 and JCM5805, as a feed additive at 108 CFU/g feed, could improve intestinal morphology, enhance immune status and disease resistance, and affect the gut microbiota of tilapia; thus, these additives could be used as probiotics for juvenile Nile tilapia. JCM5805 was more effective than JCM1136 or the mixture of the two for promoting the growth, enhancing the immune status and disease resistance of tilapia.

Calcitriol-mediated reduction in IFN-γ output in T cell large granular lymphocytic leukemia requires vitamin D receptor upregulation

Constitutively activated STAT1 and elevated IFN-γ are both characteristic of T cell large granular lymphocytic leukemia (T-LGLL), a rare incurable leukemia with clonal expansion of cytotoxic T cells due to defective apoptosis. Interferon gamma (IFN-γ) is an inflammatory cytokine that correlates with worse progression and symptomology in multiple autoimmune diseases and cancers. In canonical IFN-γ-STAT1 signaling, IFN-γ activates STAT1, a transcription factor, via phosphorylation of tyrosine residue 701 (p-STAT1). p-STAT1 then promotes transcription of IFN-γ, creating a positive feedback loop. We previously found that calcitriol treatment of the TL-1 cell line, a model of T-LGLL, significantly decreased IFN-γ secretion and p-STAT1 while increasing the vitamin D receptor (VDR) protein. Here we further explore these observations. Using TL-1 cells, IFN-γ decreased starting at 4h following calcitriol treatment, with a reduction in the intracellular and secreted protein levels as well as the mRNA content. A similar reduction in IFN-γ transcript levels was observed in primary T-LGLL patient peripheral blood mononuclear cells (PBMCs). p-STAT1 inhibition followed a similar temporal pattern and VDR upregulation inversely correlated with IFN-γ levels. Using EB1089 and 25(OH)D3, which have high or low affinity for VDR, respectively, we found that the decrease in IFN-γ correlated with the ability of EB1089, but not 25(OH)D3, to upregulate VDR. However, both compounds inhibited p-STAT1; thus the reduction of p-STAT1 is not solely responsible for IFN-γ inhibition. Conversely, cells treated with VDR siRNA exhibited decreased basal IFN-γ production upon VDR knockdown in a dose-dependent manner. Calcitriol treatment upregulated VDR and decreased IFN-γ regardless of initial VDR knockdown efficiency, strengthening the connection between VDR upregulation and IFN-γ reduction. Our findings suggest multiple opportunities to further explore the clinical relevance of the vitamin D pathway and the potential role for vitamin D supplementation in T-LGLL.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility.Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-β1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01).To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Erythropoietin treatment in murine multiple myeloma: immune gain and bone loss.

Multiple myeloma (MM) is a plasma cell malignancy, characterized by osteolytic lesions and monoclonal immunoglobulins. The anemia, accompanying the disease is often treated with recombinant human EPO. Diverse non-erythropoietic effects of EPO have led us to question its combined action on the immune system and bone in the 5T33MM mouse model. EPO administration to MM mice attenuated disease progression as demonstrated by a decrease in serum MM IgG2b, splenic CD138 expressing cells, IL-6 and RORγτ transcripts in bone marrow (BM). IFN-γ transcript levels and macrophages (F4/80+CD11b+) in the BM both increased ~1.5 fold in the EPO-treated MM mice. In-vitro, EPO stimulated phagocytosis of 5T33MM cells (+30%) by BM-derived macrophages. In contrast, high-resolution microCT analysis of distal femurs revealed EPO-associated bone loss in both healthy and 5T33MM mice. EPO significantly increased expression of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in healthy mice, but not in MM mice, likely due to antagonizing effects on MM progression. Thus, in MM, EPO may act as a double-edged-sword stimulating immune response, while accelerating bone resorption, possibly via direct action on BM macrophages. This study supports a prudent approach of treating anemia in MM patients, aiming to maintain EPO-associated anti-MM effects, while considering bone damage.

Upregulation of duck interleukin-17A during Riemerella anatipestifer infection

Although IL-17 cytokines play critical roles in host defense immunity, dysregulated expression of these cytokines is associated with inflammation and autoimmune diseases. Riemerella anatipestifer is the most important infectious bacterium in the duck industry. Interestingly, not all avian species are equally susceptible to R. anatipestifer infection. This paper reports the first description of mortality rate, bacterial burden, and expression profiles of immune-related genes between ducks and chickens infected with R. anatipestifer. Ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Comparative expression analyses of immune-related genes in R. anatipestifer-infected tissues obtained from both species revealed that TLR3, TLR7, IL-2, IL-4, and IFN-γ transcript levels were higher in chickens, whereas TLR4 and IL-17A transcript levels were higher in ducks. Marked increases in expression of IL-17A and IL-6, but not TGF-β, were associated with Th17 cell differentiation in duck splenic lymphocytes, but not in chicken splenic lymphocytes, stimulated with R. anatipestifer. Moreover, upregulation of IL-1β, IL-6, and IL-17A mRNA expressions, but not TGF-β, was confirmed in the liver and spleen of ducks infected with R. anatipestifer, indicating that IL-17A is strongly associated with Riemerella infection in ducks.

Interaction of a live attenuated Salmonella Gallinarum vaccine candidate with chicken bone marrow-derived dendritic cells

ABSTRACTSalmonella enterica serovar Gallinarum (SG) is a Gram-negative intracellular host-adapted pathogen that causes fowl typhoid. Attenuated strains of SG are proven and widely used vaccine candidates because of advantages like induction of strong humoral and cell-mediated immune responses. In the present study, we investigated the interaction of chicken bone marrow-derived dendritic cells (chBM-DCs) with an attenuated SG (JOL1355) strain that secretes a heat-labile enterotoxin B subunit protein previously shown to successfully vaccinate chickens. ChBM-DCs were isolated and cultured in the presence of recombinant chicken GM-CSF and IL-4 cytokines. The chBM-DCs were infected with JOL1355 at an multiplicity of infection of 10. JOL1355 was able to invade dendritic cells (DCs); however, the survival of JOL1355 in DCs decreased over time. At 24 h post infection, IL-6, IL-10 and IFN-γ transcript levels were significantly increased in JOL1355-infected DCs compared to non-stimulated DCs. Flow cytometry analysis showed an increased proportion of cells producing CD40, CD80, and MHC class II in the JOL1355-infected cultures compared to the non-stimulated control. In addition, JOL1355-stimulated chBM-DCs could induce significant expression of IL-2 in co-culture with autologous CD4+ T cells. Based on these results, we conclude that chBM-DCs are capable of internalizing the live attenuated SG vaccine candidate and the infected chBM-DCs show signs of maturation as evidenced by the upregulated expression of costimulatory molecules and cytokines.

Immunogenicity of Hypothetical Highly Conserved Proteins as Novel Antigens in Anaplasma marginale

Anaplasma marginale is a tick-transmitted Gram-negative intraerythrocytic bacterium and the etiological agent of bovine Anaplasmosis. Even though considerable research efforts have been undertaken, Anaplasmosis vaccine development remains a challenging field. Outer-membrane-specific antigens responsible for the ability of more complex immunogens could have a significant role in the protective response. Thus, the identification of outer-membrane antigens represents a major goal in the development of bacterial vaccines. Considering that 40% of the annotated proteins in A. marginale remain as hypothetical, we selected three candidate antigens, AM1108, AM127, and AM216 based on experimental evidence, in silico structure prediction of β-barrel outer membrane, and orthology clustering. Sequence alignment and analysis demonstrated a high degree of conservation for the three proteins between the isolates from Argentina compared to the American strains. We confirmed the transcription of the three genes in the intraerythrocytic stage. AM1108 and AM216 recombinant proteins elicited specific T-cell response proliferation and a significant rise in TNF-α and IFN-γ transcript levels, respectively. Only AM1108 was able to be recognized by specific antibodies from infected bovines. This study allowed the identification of new candidate components of the outer-membrane fraction of A. marginale. Further studies will be required to analyze their potential as effective antigens for being included in rational vaccine strategies.

Altered IL-10 and TNF-α production in peripheral blood mononuclear cells of systemic lupus erythematosus patients after Toll-like receptor 2, 4, or 9 activation

Toll-like receptor (TLR) activation and cytokines have been linked to the disease flare of systemic lupus erythematosus (SLE), yet the expression profiles of TLRs and cytokines in response to TLR activation in SLE patients remain unclear. In this study, we evaluated the expression levels of IL-10, TNF-α, interferon-γ (IFN-γ), TLR-2, TLR-4, and TLR-9 in peripheral blood mononuclear cells (PBMCs) from SLE patients and normal controls after PBMCs were stimulated with a TLR-2, TLR-4, or TLR-9 agonist. The expression levels in SLE patient group were statistically compared with those in normal control group. It was found in SLE patients that the IL-10 protein production was down-regulated after the activation of TLR-2, TLR-4, or TLR-9 and that the TNF-α protein production was decreased after the activation of TLR-2 or TLR-9, but not TLR-4. However, the transcript levels of IL-10 and TNF-α as well as the protein and transcript levels of IFN-γ were comparable between SLE and normal control groups. In addition, the TLR-2 transcript levels seem to be diminished after the activation of TLR-2, TLR-4, or TLR-9, but TLR-4 and TLR-9 transcript levels were not altered. The results indicate that the cytokine production from PBMCs in response to TLR activation is dysregulated in SLE patients, supporting the possibility that TLR activation may influence lupus disease activity through regulating cytokine production.

Invariant NKT Cells Producing IL-4 or IL-10, But Not IFN-γ, Inhibit the Th1 Response in Experimental Autoimmune Encephalomyelitis, Whereas None of These Cells Inhibits the Th17 Response

Experimental autoimmune encephalomyelitis (EAE) is mediated by Th1 and Th17 cells. Invariant NKT (iNKT) cells prevent EAE in an IL-4-, IL-10-, and IFN-γ-dependent manner. However, which of the iNKT cell-produced cytokines regulates the Th1 or Th17 response in EAE remains unclear. Wild-type B6 and Jα18(-/-) mice were immunized with MOG(35-55) peptide to address this issue. Clinical scores for EAE, IL-17, and IFN-γ transcript levels, and IL-17- or IFN-γ-expressing CD4(+) T cell percentages in the CNS and draining lymph nodes were higher in Jα18(-/-) than in B6 mice, but all of these parameters in the CNS were reduced by the adoptive transfer of wild-type or IFN-γ-deficient iNKT cells into the Jα18(-/-) mice before immunization. In contrast, adoptive transfer of IL-4- or IL-10-deficient iNKT cells into Jα18(-/-) mice decreased IL-17 transcript levels and the percentage of IL-17-expressing CD4(+) T cells in the CNS but did not affect clinical scores, IFN-γ transcript levels, or the percentage of IFN-γ-expressing CD4(+) T cells in the CNS. Taken together, IL-4- and IL-10-producing iNKT cells inhibit the Th1 cell response, but not the Th17 cell response, although wild-type iNKT cells suppress both the Th1 and Th17 responses in the CNS during EAE. Moreover, IFN-γ-producing iNKT cells have a minimal role in the regulation of the Th1 and Th17 responses in EAE.

Pronounced Virus-Dependent Activation Drives Exhaustion but Sustains IFN-γ Transcript Levels

During many chronic infections, the responding CD8 T cells become exhausted as they progressively lose their ability to elaborate key effector functions. Unlike prototypic memory CD8 cells, which rapidly synthesize IFN-gamma following activation, severely exhausted T cells fail to produce this effector molecule. Nevertheless, the ontogeny of exhausted CD8 T cells, as well as the underlying mechanisms that account for their functional inactivation, remains ill defined. We have used cytokine reporter mice, which mark the transcription of IFN-gamma mRNA by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that virus-specific CD8 T cells clearly respond during the early stages of chronic lymphocytic choriomeningitis virus infection, and that this early T cell response is more pronounced than that initially observed in acutely infected hosts. Thus, exhausted CD8 T cells appear to emerge from populations of potently activated precursors. Unlike acute infections, which result in massive expansion of the responding T cells, there is a rapid attenuation of further expansion during chronic infections. The exhausted T cells that subsequently emerge in chronically infected hosts are incapable of producing the IFN-gamma protein. Surprisingly, high levels of the IFN-gamma transcript are still present in exhausted cells, demonstrating that ablation of IFN-gamma production by exhausted cells is not due to transcriptional silencing. Thus, posttranscription regulatory mechanisms likely disable this effector module.

Bacillus subtilis spores reduce susceptibility to Citrobacter rodentium-mediated enteropathy in a mouse model

The present work was aimed at investigating whether Bacillus subtilis spores, widely used in probiotic as well as pharmaceutical preparations for mild gastrointestinal disorders, can suppress enteric infections. To address this issue, we developed a mouse model of infection using the mouse enteropathogen Citrobacter rodentium, a member of a family of human and animal pathogens which includes the clinically significant enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli strains. This group of pathogens causes transmissible colonic hyperplasia by using attaching and effacing (A/E) lesions to colonize the host colon. Because of its similarities to human enteropathogens, C. rodentium is now widely used as an in vivo model for gastrointestinal infections. Swiss NIH mice were orally administered B. subtilis spores one day before infection with C. rodentium. Mice were sacrificed on day 15 after infection, and distal colon, liver and mesenteric lymph nodes were removed for bacteria counts, morphology, immunohistology and IFNγ mRNA analysis. We observed that spore predosing was effective in significantly decreasing infection and enteropathy in suckling mice infected with a dose of C. rodentium sufficient to cause colon colonization, crypt hyperplasia and high mortality rates. Moreover, in mice predosed with spores, the number of CD4 + cells and IFNγ transcript levels remained high. These results thus indicate that our newly established model of C. rodentium infection is a suitable system for analyzing the effects of probiotic bacteria on enteroinfections and that B. subtilis spores are efficient at reducing C. rodentium infection in mice, leaving unaltered the immune response against the pathogen.

Temporal cytokine gene expression patterns in subjects with trachoma identify distinct conjunctival responses associated with infection

Ocular chlamydial disease is clinically diagnosed by the appearance of characteristic inflammatory changes and development of lymphoid follicles in the conjunctiva. Nucleic acid amplification tests and relatively non-invasive methods of sampling the conjunctival surface can be used to quantify the expression of chlamydial and host genes. Using quantitative real-time polymerase chain reaction to detect the presence of Chlamydia trachomatis (CT) 16S rRNA and human interleukin (IL)-1beta, IL-10, IL-12p40, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha transcripts we examined the immune response at the conjunctival surface in a cohort of children living in a trachoma-endemic village in The Gambia. Elevated cytokine transcript levels were associated with the presence of CT 16S rRNA. Subclinical infection (CT infection without clinical signs of disease) elicited an immune response that is proinflammatory in nature, with elevations in the transcription of IL-1beta, IFN-gamma and IL-12p40. Clinically apparent infections were associated with the elevation of mRNA for the multi-functional cytokine TNF-alpha (fibrotic, type 1 inflammatory and regulatory) and the counter regulatory cytokine, IL-10, in addition to the other proinflammatory cytokines. A positive correlation between IFN-gamma transcript levels and the amount of CT 16S rRNA expressed in conjunctiva was found.