IL-10 Production In Response Research Articles

Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production.

Polymorphonuclear leukocytes (PMNs) are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the antimicrobial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its antimicrobial activity by inhibiting IL-10 production. PMNs from wild-type mice did not increase IL-10 production in response to S. pneumoniae; however, CD73-/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of WT PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73-/- PMNs as well as host resistance of CD73-/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake, and intracellular killing or production of antimicrobial neutrophil elastase and myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal reactive oxygen species (ROS) production by PMNs. ROS contributed to PMN antimicrobial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN antimicrobial phenotype during S. pneumoniae infection.

Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in β-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFβ and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.

Expanding Clinical Phenotype and Novel Insights into the Pathogenesis of ICOS Deficiency

BackgroundInducible T cell co-stimulator (ICOS) deficiency has been categorized as a combined immunodeficiency often complicated by enteropathies, autoimmunity, lymphoproliferation, and malignancy. We report seven new patients and four novel ICOS mutations resulting in a common variable immunodeficiency (CVID)–like phenotype and show that dysregulated IL-12 release, reduced cytotoxic T lymphocyte–associated protein 4 (CTLA4) expression, and skewing towards a Th1-dominant phenotype are all associated with inflammatory complications in this condition.MethodsA combination of whole exome and Sanger sequencing was used to identify novel mutations. Standard clinical and immunological evaluation was performed. FACS and ELISA-based assays were used to study cytokine responses and ICOS/ICOSL/CTLA4 expression following stimulation of whole blood and PBMCs with multiple TLR ligands, anti-CD3, and PHA.ResultsFour novel ICOS mutations included homozygous c.323_332del, homozygous c.451C>G, and compound heterozygous c.58+1G>A/c.356T>C. The predominant clinical phenotype was that of antibody deficiency associated with inflammatory complications in 4/7 patients. Six out of seven patients were treated with immunoglobulin replacement and one patient died from salmonella sepsis. All patients who were tested showed reduced IL-10 and IL-17 cytokine responses, normal IL-1β, IL6, and TNF release following LPS stimulation and highly elevated IL-12 production in response to combined LPS/IFNγ stimulation. This was associated with skewing of CD4+ T cells towards Th1 phenotype and increased expression of ICOSL on monocytes. Lastly, reduced CTLA4 expression was found in 2 patients. One patient treated with ustekinumab for pancytopenia due to granulomatous bone marrow infiltration failed to respond to this targeted therapy.ConclusionsICOS deficiency is associated with defective T cell activation, with simultaneously enhanced stimulation of monocytes. The latter is likely to result from a lack of ICOS/ICOSL interaction which might be necessary to provide negative feedback which limits monocytes activation.

CUL4B negatively regulates Toll-like receptor-triggered proinflammatory responses by repressing Pten transcription

Toll-like receptors (TLRs) play critical roles in innate immunity and inflammation. The molecular mechanisms by which TLR signaling is fine-tuned remain to be completely elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING E3 ligase complex (CRL4B), has been shown to regulate diverse developmental and physiological processes by catalyzing monoubiquitination for histone modification or polyubiquitination for proteasomal degradation. Here, we identified the role of CUL4B as an intrinsic negative regulator of the TLR-triggered inflammatory response. Deletion of CUL4B in macrophages increased the production of proinflammatory cytokines and decreased anti-inflammatory cytokine IL-10 production in response to pathogens that activate TLR3, TLR4, or TLR2. Myeloid cell-specific Cul4b knockout mice were more susceptible to septic shock when challenged with lipopolysaccharide, polyinosinic-polycytidylic acid or Salmonella typhimurium infection. We further demonstrated that enhanced TLR-induced inflammatory responses in the absence of CUL4B were mediated by increased GSK3β activity. Suppression of GSK3β activity efficiently blocked the TLR-triggered increase in proinflammatory cytokine production and attenuated TLR-triggered death in Cul4b mutant mice. Mechanistically, CUL4B was found to negatively regulate TLR-triggered signaling by epigenetically repressing the transcription of Pten, thus maintaining the anti-inflammatory PI3K-AKT-GSK3β pathway. The upregulation of PTEN caused by CUL4B deletion led to uncontrolled GSK3β activity and excessive inflammatory immune responses. Thus, our findings indicate that CUL4B functions to restrict TLR-triggered inflammatory responses through regulating the AKT-GSK3β pathway.

Single cell analysis reveals immune cell-adipocyte crosstalk regulating the transcription of thermogenic adipocytes.

Immune cells are vital constituents of the adipose microenvironment that influence both local and systemic lipid metabolism. Mice lacking IL10 have enhanced thermogenesis, but the roles of specific cell types in the metabolic response to IL10 remain to be defined. We demonstrate here that selective loss of IL10 receptor α in adipocytes recapitulates the beneficial effects of global IL10 deletion, and that local crosstalk between IL10-producing immune cells and adipocytes is a determinant of thermogenesis and systemic energy balance. Single Nuclei Adipocyte RNA-sequencing (SNAP-seq) of subcutaneous adipose tissue defined a metabolically-active mature adipocyte subtype characterized by robust expression of genes involved in thermogenesis whose transcriptome was selectively responsive to IL10Rα deletion. Furthermore, single-cell transcriptomic analysis of adipose stromal populations identified lymphocytes as a key source of IL10 production in response to thermogenic stimuli. These findings implicate adaptive immune cell-adipocyte communication in the maintenance of adipose subtype identity and function.

ADAM17-Mediated Reduction in CD14++CD16+ Monocytes ex vivo and Reduction in Intermediate Monocytes With Immune Paresis in Acute Pancreatitis and Acute Alcoholic Hepatitis.

Impaired immune responses and increased susceptibility to infection characterize acute inflammatory conditions such as pancreatitis and alcoholic hepatitis and are major causes of morbidity and mortality. However, the mechanisms that drive this apparent immune paresis remain poorly understood. Monocytes mediate host responses to damage and pathogens in health and disease, and three subsets of monocytes have been defined based on CD14 and CD16 expression. We sought to determine the changes in monocyte subsets in acute pancreatitis (AP) and acute alcoholic hepatitis (AAH), together with functional consequences and mechanisms that underlie this change. Peripheral blood mononuclear cells (PBMCs) from patients with AP or AAH were compared with healthy controls. Monocyte subsets were defined by HLA-DR, CD14, and CD16 expression. Changes in surface and intracellular protein expression and phosphorylation were determined by flow cytometry. Phenotype and function were assessed following stimulation with lipopolysaccharide (LPS) or other agonists in the presence of specific inhibitors of TNFα and a disintegrin and metalloproteinase 17 (ADAM17). Patients with AP and AAH had reduced CD14++CD16+ intermediate monocytes compared to controls. Reduction of intermediate monocytes was recapitulated ex vivo by stimulating healthy control PBMCs with Toll-like receptor (TLR) agonists LPS, flagellin or polyinosilic:polycytidylic acid (poly I:C). Stimulation caused shedding of CD14 and CD16, which could be reversed using the ADAM17 inhibitor, TMI005 but not direct inhibitors of TNFα, a known ADAM17-target. Culturing PBMCs from healthy controls resulted in expansion of intermediate monocytes, which did not occur when LPS was in the culture medium. Cultured intermediate monocytes showed reduced expression of CX3CR1, CCR2, TLR4, and TLR5. We found reduced migratory responses, intracellular signaling and pro-inflammatory cytokine production, and increased expression of IL-10. Stimulation with TLR agonists results in ADAM17-mediated shedding of phenotypic markers from CD16+ monocytes, leading to apparent “loss” of intermediate monocytes. Reduction in CD14++CD16− monocytes and increased CD14++CD16+ is associated with altered responses in functional assays ex vivo. Patients with AP and AAH had reduced proportions of CD14++CD16+ monocytes and reduced phosphorylation of NFκB and IL-6 production in response to bacterial LPS. Together, these processes may contribute to the susceptibility to infection observed in AP and AAH.

MEK2 Negatively Regulates Lipopolysaccharide-Mediated IL-1β Production through HIF-1α Expression.

LPS-activated macrophages require metabolic reprogramming and glucose uptake mediated by hypoxia-inducible factor (HIF)-1 α and glucose transporter 1 (Glut1) expression for proinflammatory cytokine production, especially IL-1β. This process is tightly regulated through activation of MAPK kinases, including the MEK/ERK pathway as well as several transcription factors including HIF-1α. Although MAPK kinase (MEK) 2 deficiency had no significant effect on NO, TNF-α, or IL-12 production in response to LPS challenge, MEK2-deficient murine bone marrow-derived macrophages (BMDMs) exhibited lower IL-10 production. Importantly, MEK2-deficient BMDMs exhibited a preserved ERK1/2 phosphorylation, higher HIF-1α and Glut1 levels, and substantially increased IL-1β as well as IL-6 production in response to LPS stimulation. Knockdown of HIF-1α expression via short interference RNA decreased the level of HIF-1α expression in MEK2-deficient BMDMs and decreased IL-1β production in response to LPS treatment. Furthermore, we performed gain of function experiments by overexpressing MEK2 protein in RAW264.7 cells. LPS stimulation of MEK2 overexpressed in RAW264.7 cells led to a marked decreased IL-1β production. Finally, we investigated the role of Mek1 and Mek2 double and triple mutation on ERK phosphorylation, HIF-1α expression, and IL-1β production. We found that MEK2 is the major kinase, which inversely proportionally regulates HIF-1α and IL-1β expression independent of ERK activation. Our findings demonstrate a novel regulatory function for MEK2 in response to TLR4 activation in IL-1β production through modulating HIF-1α expression.

Loss of RNA-binding protein GRSF1 activates mTOR to elicit a proinflammatory transcriptional program.

The RNA-binding protein GRSF1 (G-rich RNA sequence-binding factor 1) critically maintains mitochondrial homeostasis. Accordingly, loss of GRSF1 impaired mitochondrial respiration and increased the levels of reactive oxygen species (ROS), triggering DNA damage, growth suppression, and a senescent phenotype characterized by elevated production and secretion of interleukin (IL)6. Here, we characterize the pathways that govern IL6 production in response to mitochondrial dysfunction in GRSF1-depleted cells. We report that loss of GRSF1 broadly altered protein expression programs, impairing the function of respiratory complexes I and IV. The rise in oxidative stress led to increased DNA damage and activation of mTOR, which in turn activated NF-κB to induce IL6 gene transcription and orchestrate a pro-inflammatory program. Collectively, our results indicate that GRSF1 helps preserve mitochondrial homeostasis, in turn preventing oxidative DNA damage and the activation of mTOR and NF-κB, and suppressing a transcriptional pro-inflammatory program leading to increased IL6 production.

Acanthamoeba proteases contribute to macrophage activation through PAR1 , but not PAR2.

Acanthamoeba infections are characterized by an intense localized innate immune response associated with an influx of macrophages. Acanthamoeba protease production is known to affect virulence. Herein, the ability of Acanthamoeba trophozoite proteases, of either the laboratory Neff strain or a recently isolated clinical strain, to stimulate IL-12 and IL-6 and to activate protease-activated receptors, PAR1 and PAR2 expressed on murine macrophages, was investigated. Using selected protease inhibitors, leupeptin and E64, we showed that Acanthamoeba proteases can stimulate IL-12 and IL-6 by murine macrophages. Subsequently, using specific antagonists to inhibit PAR1 , and bone marrow-derived macrophages from PAR2 gene-deficient mice, we demonstrate that PAR1 , but not PAR2 contributes to macrophage IL-12 production in response to Acanthamoeba. In contrast, Acanthamoeba-induced IL-6 production is PAR1 and PAR2 independent. This study shows for the first time the involvement of PARs, expressed on macrophages, in the response to Acanthamoeba trophozoites and might provide useful insight into Acanthamoeba infections and their future treatments.

Analyses of Foxp3+ Treg cells and Tr1 cells in subcutaneous immunotherapy-treated allergic individuals in humans and mice

Subcutaneous immunotherapy (SCIT) is a causative treatment for allergic diseases. More recently, it has become clear that regulatory T (Treg) cells are increased by SCIT. Treg cells are generally divided into two main groups: 1) CD25+ Foxp3+ CD4+ T cells (Foxp3+ Treg cells) and 2) IL-10-producing Foxp3- CD4+ T cells (Tr1 cells). We demonstrated that the number of Tr1 cells in peripheral blood mononuclear cells in SCIT-treated pollinosis patients were significantly higher than that in non-SCIT-treated patients, but Foxp3+ Treg cells were not. Consistent with the results of human peripheral blood, Tr1 cells were increased in the lungs of asthmatic mice by SCIT, but Foxp3+ Treg cells were not. Moreover, in vitro-induced Tr1 cells were responded to the antigen to produce a large amount of IL-10 in in vitro and in vivo. Adoptive transfer of the induced Tr1 cells significantly suppressed the development of asthma. In any species of human and mouse, the increase in Tr1 cells rather than Foxp3+ Treg cells could be important for the effects of SCIT. The increased Tr1 cells by SCIT functionally suppressed allergic asthma probably via production of IL-10 in response to the specific antigen. Therefore, analyses of the induction mechanisms of Tr1 cells and search for compounds which induce Tr1 cells are thought to lead to development of more efficient SCIT.

Comparative efficacy of two heterologous capripox vaccines to control lumpy skin disease in cattle

This longitudinal study was performed for field trial of heterologous vaccine strains of lumpy skin disease (LSD) to provide details on the characteristics of induced immune responses by measurement of specific antibody and target cytokines – critical parameters in immune response that can be related to the durability of protection. The experimental calves were vaccinated with Gorgan-GPV and RM/65-SPV vaccines and humoral and cellular immunity were evaluated weekly. In each vaccinated groups, cross-neutralising antibody titers against LSD virus (LSDV) could be detected, and this rate in GGPV-vaccinated calves (GC) was higher than RSPV-vaccinated calves (RC) in all weeks of experiments. The stimulation index and IFN-γ and IL-4 production in response to homologous virus were higher than to the heterologous virus in all time points. The highest difference between them was observed in RVC, and a significant difference were only shown at 21-day post vaccination (DPV) (p<0.001). The results of this study indicated that GGPV-vaccine had a good immunogenic response due to induction of high antibody titre and higher lymphocyte proliferation and IFN-γ and IL-4 production. Therefore, it was considered suitable to control LSD.

Myeloma Cells Have the Ability to Suppress Th1-Inducing Capacity but Enhance Th2-Mediated Response of Dendritic Cells By Producing Factors Other Than Soluble SLAMF7

Background: Lenalidomide (LEN), as a backbone drug for multiple myeloma (MM), has both direct tumoricidal and immunomodulatory effects in MM. LEN displays immunopotentiating activity, including augmentation of T and natural killer cell function and to induce selective reduction of regulatory T cells. Thus, the immune-based cancer treatments are currently important in MM therapy. Since myeloma cells together with immune cells are immersed in the pathogenic micro-environment, understanding the influence of micro-environment against myeloma cells and immune system may lead to the development of novel treatment strategies for MM. Series of analyses have clarified a functional plasticity of dendritic cells (DCs) to induce Th1 or Th2 response and DCs are pivotal in orchestrating both innate and acquire immune responses also in the anti-tumor immunity. Here, we focused on the effects of myeloma environment to modulate the human myeloid DCs (mDCs), which are the major regulators to induce Th1 or Th2 responses. Soluble form of SLAMF7 (sSLAMF7) is a pivotal environmental component of MM and functions as a self-ligand of myeloma cells. sSLAMF7 has been detected in the serum of patients with MM at higher levels than healthy individuals and therefore may presumably play some roles in pathophysiological condition of MM. To evaluate the functions of soluble factors/components derived from myeloma cells, we analyzed whether supernatants of myeloma cell lines could modulate mDC functions. Methods: Using cell sorting, blood human CD11+ mDCs were isolated from healthy adult volunteers. The DC functions were analyzed after culture for 24 h with supernatant of myeloma cell lines, NCI-H929 (H929) or MM.1R. This study was approved by the Institutional Review Board of Kansai Medical University.Results: We here found that both supernatants of myeloma cell lines, H929 and MM.1R did not affect CD86 expression on mDCs in response to toll-like receptor (TLR)-ligand (R848), but upregulated their CD86 expression in response to Th2-inducing cytokine, thymic stromal lymphopoietin (TSLP). Of note, the supernatants inhibited mDC-derived IL-12 production in response to R848 (p < 0.05: H929; 25-30% of cont. and MM1.R; less than 50 % of cont.), while in response to TSLP, dose-dependently enhanced the Th2-recruiting chemokine CCL17/TARC production (p < 0.05: H929; 130- 210 % of cont. and MM1.R; 120-185 % of cont.), which functions as chemoattractant for memory Th2 cells and contribute to promote humoral response. In addition, we revealed that these capacities were further enhanced by the addition of LEN at clinical in vivo plasma concentration of 1 µM. Thus, MM micro-environment could have the potential to shift DC-mediated response from Th1 to Th2. To clarify whether sSLAMF7 in the supernatant of myeloma cell lines play a role in the Th1/Th2-modulating effect, we calculated the sSLAMF7 concentration in the supernatants and analyzed an inhibitory effect of elotuzumab for the DC function induced by the supernatants. sSLAMF7 could not be detected at all in the supernatants of both myeloma cell lines, even in any concentration of cells. Furthermore, addition of 30 to 100 ug/ml (clinical trough serum concentration) elotuzumab into the culture of mDCs and the supernatants did not affect the CD86 expression and IL-12/CCL17 production. Hence, factors other than sSLAMF7 from myeloma cells can trigger DCs to suppress Th1 response and to promote Th2 response.Conclusion: Our data suggest that the MM micro-environment, while suppress the Th1-inducing capacity, rather lead to enhance Th2 response at DC phase. As sSLAMF7 is detected in the serum of patients with MM but not in the supernatants of MM cell lines in the experimental setting, there is a limitation that our data does not consider the action of sSLAMF7. However, the present result provides an important clue for understanding the biological basis for the MM micro-environment against host immune defense at onset. DisclosuresIto:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria; Mundipharma: Honoraria; Celgene: Honoraria.

1532. Increased Risk of Bacterial, Fungal and Other Viral Infections During CMV Infection: Decreased Cytokine Production in Response to Toll-Like Receptor Ligands

BackgroundIn the solid-organ transplant (SOT) setting, CMV is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal and viral infections. However, the pathogenesis of this phenomenon is poorly understood. The aim of our study was to determine whether inflammatory responses to different Toll-like receptor ligands are blunted during CMV infection in SOT patients.MethodsCMV D+/R− SOT patients had blood drawn at the end of CMV prophylaxis and then weekly after onset of CMV viremia. PBMCs were extracted and incubated for 24 hours in the presence of bacterial (LPS), fungal (Zymosan [ZYM]), and viral (Resiquimod [R848]) ligands. Proinflammatory (IL1β), Th1 (IFNγ), Th2 (IL4), immunoregulatory (IL10), and chemotactic (MCP1) cytokines were measured in the supernatant by multiplex ELISA.ResultsThirty-eight SOT patients were followed for at least 9 months. Patients who developed subsequent CMV infection had lower cytokines in response to bacterial, fungal and viral ligands (LPS, ZYM, and R848) at the end of prophylaxis compared with those with no CMV infection. These results were independent of immunosuppression and peripheral blood cell counts. Specifically, these trends were significantly different with respect to IFNγ, IL1β, and IL10 production in response to LPS (P = 0.003, 0.003, and 0.039, respectively), R848 (P < 0.001, 0.039, and <0.001, respectively) and ZYM (P = 0.039, 0.003, and 0.003, respectively), as well as for MCP1 in response to R848 or ZYM (P = 0.039 for both). In the cohort with CMV infection, cytokine responses to TLR ligands were even lower during the acute CMV infection when compared with the end of prophylaxis, although this was significant only for IL10 production after R848 stimulation (P = 0.034). There was no influence of CMV viral load or duration of viremia on cytokine levels.ConclusionResponse to non-CMV antigens during CMV infection was blunted supporting the clinical observation in transplant recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. However, inherent differences in patients that are neither directly related to CMV nor to their net level of immunosuppression also contribute to this increased susceptibility, as cytokine levels at the end of prophylaxis were lower among patients with compared with those without subsequent CMV infection.Disclosures All authors: No reported disclosures.

Promoting cytokine production

The helicase DHX9 is required for production of the cytokine IL-6 in response to Toll-like receptor stimulation.

Low Interleukin 10 Production at Birth Is a Risk Factor for Atopic Dermatitis in Neonates with Bifidobacterium Colonization

Background: Altered regulatory immune responses to microbial stimuli and intestinal colonization of beneficial bacteria early in life may contribute to the development of allergic diseases (e.g., atopic dermatitis [AD]). However, few reports have investigated these factors simultaneously. The purpose of this study was to analyze neonatal immune responses to microbial stimuli as well as intestinal colonization of beneficial bacteria, in relation to the development of AD in a birth cohort. Methods: Pregnant women were recruited, and their infants were followed up until 7 months of age. Levels of interleukin (IL)-10 released from cord-blood mononuclear cells (CBMCs) stimulated with heat-killed gram-positive bacteria (Bifidobacterium bifidum and Lactobacillus rhamnosus GG) and Lactobacillus-derived peptidoglycan were measured. Fecal Bifidobacterium counts at 4 days and 1 month were quantified using real-time polymerase chain reaction. The development of AD was determined by means of a questionnaire at 7 months of age. Results: The levels of released IL-10 were significantly lower in infants with AD (n = 17) than in infants without AD (n = 53) for all stimuli. In infants with fecal Bifidobacterium, the incidence of AD was inversely associated with the release of IL-10 from cord blood mononuclear cells. Conclusion: Our findings suggest that impaired IL-10 production in response to microbial stimuli at birth may be associated with an increased risk of developing infantile AD, even in infants with early colonization of intestinal bifidobacteria.

Langerin+ CD8α+ Dendritic Cells Drive Early CD8+ T Cell Activation and IL-12 Production During Systemic Bacterial Infection.

Bloodstream infections induce considerable morbidity, high mortality, and represent a significant burden of cost in health care; however, our understanding of the immune response to bacteremia is incomplete. Langerin+ CD8α+ dendritic cells (DCs), residing in the marginal zone of the murine spleen, have the capacity to cross-prime CD8+ T cells and produce IL-12, both of which are important components of antimicrobial immunity. Accordingly, we hypothesized that this DC subset may be a key promoter of adaptive immune responses to blood-borne bacterial infections. Utilizing mice that express the diphtheria toxin receptor under control of the langerin promoter, we investigated the impact of depleting langerin+ CD8α+ DCs in a murine model of intravenous infection with Mycobacterium bovis bacille Calmette–Guerin (BCG). In the absence of langerin+ CD8α+ DCs, the immune response to blood-borne BCG infection was diminished: bacterial numbers in the spleen increased, serum IL-12p40 decreased, and delayed CD8+ T cell activation, proliferation, and IFN-γ production was evident. Our data revealed that langerin+ CD8α+ DCs play a pivotal role in initiating CD8+ T cell responses and IL-12 production in response to bacteremia and may influence the early control of systemic bacterial infections.

Variable localization of Toll-like receptors in human fallopian tube epithelial cells.

ObjectiveTo determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells.MethodsThe localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1–10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands.ResultsFallopian tube epithelial cells expressed TLRs 1–10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8.ConclusionOur findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

Association between impaired IL-10 production following exposure to Staphylococcus aureus enterotoxin B and disease severity in eosinophilic chronic rhinosinusitis

BackgroundIL-10 is a major anti-inflammatory cytokine that prevents inflammation-mediated tissue damage. We characterized the production of IL-10 by sinonasal tissue cells following exposure to Staphylococcus aureus enterotoxin B (SEB), which elicits cellular responses and is associated with the pathogenesis of eosinophilic chronic rhinosinusitis (ECRS). MethodsDispersed nasal polyp (NP) cells and uncinate tissue (UT) cells were prepared from patients with CRS with and without NP, respectively. Cells were incubated with SEB, and then the levels of IL-10 in the cell supernatants were determined. The effect of neutralizing IL-10 on SEB-induced IL-5, IL-13, IFN-γ, and IL-17A production was examined. Expression of IL-10 in NPs was also determined. ResultsIL-10 was expressed in infiltrating inflammatory cells in NPs. NP cells, especially non-adherent NP cells, produced substantial amounts of IL-10 in response to SEB. Although baseline production of IL-10 was significantly higher in NP cells than UT cells, the degree of IL-10 response to SEB was not significantly different between the cell types. The degree of IL-10 production was negatively correlated with the degree of eosinophilia both in tissues and peripheral blood whereas positively correlated with the 1-s forced expiratory volume/forced vital capacity ratio. Patients with severe ECRS displayed a significant decrease in IL-10 production compared with those with non-ECRS. IL-10 neutralization significantly augmented SEB-induced IL-13 and IFN-γ production by NP cells. ConclusionsImpaired IL-10 production in response to SEB in NP may exacerbate the pathophysiology of ECRS including eosinophilia and lower airway obstruction.

Innate immune responses following Kawasaki disease and toxic shock syndrome.

The pathogenesis of Kawasaki disease (KD) remains unknown and there is accumulating evidence for the importance of the innate immune system in initiating and mediating the host inflammatory response. We compared innate immune responses in KD and toxic shock syndrome (TSS) participants more than two years after their acute illness with control participants to investigate differences in their immune phenotype. Toxic shock syndrome shares many clinical features with KD; by including both disease groups we endeavoured to explore changes in innate immune responses following acute inflammatory illnesses more broadly. We measured the in vitro production of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-1 receptor antagonist (IL-1ra), and IL-10 following whole blood stimulation with toll-like receptor and inflammasome ligands in 52 KD, 20 TSS, and 53 control participants in a case-control study. Analyses were adjusted for age, sex, and unstimulated cytokine concentrations. Compared to controls, KD participants have reduced IL-1ra production in response to stimulation with double stranded RNA (geometric mean ratio (GMR) 0.37, 95% CI 0.15, 0.89, p = 0.03) and increased IL-6 production in response to incubation with Lyovec™ (GMR 5.48, 95% CI 1.77, 16.98, p = 0.004). Compared to controls, TSS participants have increased IFN-γ production in response to peptidoglycan (GMR 4.07, 95% CI 1.82, 9.11, p = 0.001), increased IL-1β production to lipopolysaccharide (GMR 1.64, 95% CI 1.13, 2.38, p = 0.01) and peptidoglycan (GMR 1.61, 95% CI 1.11, 2.33, p = 0.01), and increased IL-6 production to peptidoglycan (GMR 1.45, 95% CI 1.10, 1.92, p = 0.01). Years following the acute illness, individuals with previous KD or TSS exhibit a pro-inflammatory innate immune phenotype suggesting a possible underlying immunological susceptibility or innate immune memory.

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita, western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8+ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.