IL-10 Production In Response Research Articles

Intravenous immunoglobulin skews macrophages to an anti-inflammatory, IL-10-producing activation state

Intravenous Ig is used to treat autoimmune or autoinflammatory disorders, but the mechanism by which it exerts its immunosuppressive activity is not understood completely. To examine the impact of intravenous Ig on macrophages, we compared cytokine production by LPS-activated macrophages in the presence and absence of intravenous Ig. Intravenous Ig treatment induced robust production of IL-10 in response to LPS, relative to LPS stimulation alone, and reduced production of proinflammatory cytokines. This anti-inflammatory, intravenous Ig-induced activation was sustained for 24 h but could only be induced if intravenous Ig were provided within 1 h of LPS stimulation. Intravenous Ig activation led to enhanced and prolonged activation of MAPKs, Erk1/2, p38, and Erk5, and inhibition of each reduced intravenous Ig-induced IL-10 production and suppression of IL-12/23p40. IL-10 production occurred rapidly in response to intravenous Ig + LPS and was sufficient to reduce proinflammatory IL-12/23p40 production in response to LPS. IL-10 induction and reduced IL-12/23p40 production were transcriptionally regulated. IL-10 played a direct role in reducing proinflammatory cytokine production by macrophages treated with intravenous Ig + LPS, as macrophages from mice deficient in the IL-10R β chain or in IL-10 were compromised in their ability to reduce proinflammatory cytokine production. Finally, intraperitoneal injection of intravenous Ig or intravenous Ig + LPS into mice activated macrophages to produce high levels of IL-10 during subsequent or concurrent LPS challenge, respectively. These findings identify IL-10 as a key anti-inflammatory mediator produced by intravenous Ig-treated macrophages and provide insight into a novel mechanism by which intravenous Ig may dampen down inflammatory responses in patients with autoimmune or autoinflammatory diseases.

Age-associated modifications of intestinal permeability and innate immunity in human small intestine.

The physical and immunological properties of the human intestinal epithelial barrier in aging are largely unknown. Ileal biopsies from young (7-12years), adult (20-40years) and aging (67-77years) individuals not showing symptoms of gastrointestinal (GI) pathologies were used to assess levels of inflammatory cytokines, barrier integrity and cytokine production in response to microbial challenges. Increased expression of interleukin (IL)-6, but not interferon (IFN)γ, tumour necrosis factor (TNF)-α and IL-1β was observed during aging; further analysis showed that cluster of differentiation (CD)11c(+) dendritic cells (DCs) are one of the major sources of IL-6in the aging gut and expressed higher levels of CD40. Up-regulated production of IL-6 was accompanied by increased expression of claudin-2 leading to reduced transepithelial electric resistance (TEER); TEER could be restored in invitro and ex vivo cultures by neutralizing anti-IL-6 antibody. In contrast, expression of zonula occludens-1 (ZO-1), occludin and junctional-adhesion molecule-A1 did not vary with age and overall permeability to macromolecules was not affected. Finally, cytokine production in response to different microbial stimuli was assessed in a polarized invitro organ culture (IVOC). IL-8 production in response to flagellin declined progressively with age although the expression and distribution of toll-like receptor (TLR)-5 on intestinal epithelial cells (IECs) remained unchanged. Also, flagellin-induced production of IL-6 was less pronounced in aging individuals. In contrast, TNF-α production in response to probiotics (VSL#3) did not decline with age; however, in our experimental model probiotics did not down-regulate the production of IL-6 and expression of claudin-2. These data suggested that aging affects properties of the intestinal barrier likely to impact on age-associated disturbances, both locally and systemically.

Inflammatory and Immune Activation in Intestinal Myofibroblasts Is Developmentally Regulated.

We previously demonstrated that intestinal myofibroblasts from immature tissue produce excessive IL-8 in response to Escherichia coli lipopolysaccharide (LPS) compared to cells from mature tissue. However, it is unknown whether other cytokines and TLR agonists contribute to this developmentally regulated response. The aim of this study was to further characterize differences in inflammatory signaling in human primary intestinal fibroblasts from fetal (FIF) and infant (IIF) tissue and examine their potential to activate the adaptive immune response in vitro. Cytokine profiles of LPS-stimulated FIF and IIF were assessed by cytokine profile array. IL-8, IL-6, and IL-10 production in response to TLR2, TLR2/6, TLR4, and TLR5 agonists was determined by quantitative ELISA. The potential of activated myofibroblasts to activate adaptive immunity was determined by measuring surface class II MHC expression using flow cytometry. LPS-stimulated FIF produced a distinct proinflammatory cytokine profile consisting of MCP-1, GRO-alpha, IL-6, and IL-8 expression. FIF produced significant IL-8 and IL-6 in response to TLR4 agonist. IIF produced significant levels of IL-8 and IL-6 in the presence of TLR5 and TLR2 agonists. IFN-γ-treated FIF expressed greater HLA-DR levels compared to unstimulated controls and IFN-γ- and LPS-treated IIF. Activated FIF produce a more diverse inflammatory cytokine profile and greater levels of IL-8 and IL-6 in response to TLR4 stimulation compared to IIF. FIF express class II MHC proteins associated with activation of the adaptive immune response. These data suggest that FIF may contribute to bacterial-associated gut inflammation in the immature intestine.

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors.

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors.

Lack of TNFR2 expression by CD4+ T cells limits IL-2 production and promotes Th17 differentiation (BA11P.138)

Abstract Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.

Household air pollution causes dose-dependent inflammation and altered phagocytosis in human macrophages.

Three billion people are exposed to household air pollution from biomass fuel use. Exposure is associated with higher incidence of pneumonia, and possibly tuberculosis. Understanding mechanisms underlying these defects would improve preventive strategies. We used human alveolar macrophages obtained from healthy Malawian adults exposed naturally to household air pollution and compared them with human monocyte-derived macrophages exposed in vitro to respirable-sized particulates. Cellular inflammatory response was assessed by IL-6 and IL-8 production in response to particulate challenge; phagosomal function was tested by uptake and oxidation of fluorescence-labeled beads; ingestion and killing of Streptococcus pneumoniae and Mycobacterium tuberculosis were measured by microscopy and quantitative culture. Particulate ingestion was quantified by digital image analysis. We were able to reproduce the carbon loading of naturally exposed alveolar macrophages by in vitro exposure of monocyte-derived macrophages. Fine carbon black induced IL-8 release from monocyte-derived and alveolar macrophages (P < 0.05) with similar magnitude responses (log10 increases of 0.93 [SEM = 0.2] versus 0.74 [SEM = 0.19], respectively). Phagocytosis of pneumococci and mycobacteria was impaired with higher particulate loading. High particulate loading corresponded with a lower oxidative burst capacity (P = 0.0015). There was no overall effect on killing of M. tuberculosis. Alveolar macrophage function is altered by particulate loading. Our macrophage model is comparable morphologically to the in vivo uptake of particulates. Wood smoke-exposed cells demonstrate reduced phagocytosis, but unaffected mycobacterial killing, suggesting defects related to chronic wood smoke inhalation limited to specific innate immune functions.

Differential Membrane Expression of Toll-Like Receptors and Intracellular Cytokine Induction in Peripheral Blood Monocytes of Patients with Chronic Kidney Disease and Diabetic Nephropathy

Background: Toll-like receptors (TLRs) are key players in the innate immune system whose activation leads to an inflammatory response. Inflammation plays an important role in the pathogenesis of chronic kidney disease (CKD) and diabetes mellitus. The aim of our study was to assess the proinflammatory state of nondialysis CKD patients by evaluating the membrane expression of TLR2 and TLR4 and the intracellular IL-1β and IL-6 production in response to the ligand Pam3Cys-Ser-(Lys)4 (Pam3CSK4). Methods: 85 nondialysis CKD patients [mean estimated glomerular filtration rate: 34 (17-90) ml/min/1.73 m²] were divided in 2 groups: 55 nondiabetic CKD patients (CKD group) and 30 patients with diabetic nephropathy (DN group). The two groups were compared with 36 healthy subjects (control group). TLR2 and TLR4 membrane expression in monocytes and Pam3CSK4-induced intracellular production of IL-1β and IL-6 were assessed by flow cytometry. Results: Both patient groups showed increased TLR2 membrane expression compared with the control group, both at baseline (p < 0.05 for both) and after Pam3CSK4 stimulation (p < 0.05 for both). The DN group exhibited significantly higher TLR4 expression at baseline compared to the CKD and control groups (p < 0.04 and p < 0.02, respectively). Intracellular IL-1β and IL-6 levels at baseline were significantly lower in CKD patients compared to the DN and control groups. After Pam3CSK4 stimulation, intracellular IL-1β and IL-6 increased in all groups, but were lower in the CKD group versus the control group or DN group, which exhibited higher levels than the controls. Conclusions: Nondialysis CKD patients showed significant alterations in TLR2 and TLR4 membrane expression, and impaired Pam3CSK4-induced cytokine production in monocytes, a phenomenon that is markedly influenced by the presence of diabetes.

Therapeutic immunisation plus cytokine and hormone therapy improves CD4 T-cell counts, restores anti-HIV-1 responses and reduces immune activation in treated chronic HIV-1 infection

BackgroundThis randomised, open label, phase I, immunotherapeutic study investigated the effects of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human growth hormone (rhGH), and therapeutic immunisation (a Clade B DNA vaccine) on combination antiretroviral therapy (cART)-treated HIV-1-infected individuals, with the objective to reverse residual T-cell dysfunction. MethodsTwelve HIV-1+ patients on suppressive cART with baseline CD4 T-cell counts >400cells/mm3 blood were randomised into one of three groups: (1) vaccine, IL-2, GM-CSF and rhGH (n=3); (2) vaccine alone (n=4); or (3) IL-2, GM-CSF and rhGH (n=5). Samples were collected at weeks 0, 1, 2, 4, 6, 8, 12, 16, 24 and 48. Interferon (IFN)-γ, IL-2, IL-4 and perforin ELISpot assays performed at each time point quantified functional responses to Gag p17/p24, Nef, Rev, and Tat peptides; and detailed T-cell immunophenotyping was undertaken by flow cytometry. Proviral DNA was also measured. ResultsMedian baseline CD4 T-cell count was 757cells/mm3 (interquartile range [IQR] 567–886cells/mm3), median age 48 years (IQR 42–51 years), and plasma HIV-1-RNA <50copies/ml for all subjects. Patients who received vaccine plus IL-2, GM-CSF and rhGH (group 1) showed the most marked changes. Assessing mean changes from baseline to week 48 revealed significantly elevated numbers of CD4 T cells (p=0.0083) and improved CD4/CD8 T-cell ratios (p=0.0033). This was accompanied by a significant reduction in expression of CD38 on CD4 T cells (p=0.0194), significantly increased IFN-γ and IL-2 production in response to Gag (p=0.0122) and elevated IFN-γ production in response to Tat (p=0.041) at week 48 compared to baseline. Subjects in all treatment groups showed significantly reduced PD-1 expression at week 48 compared to baseline, with some reductions in proviral DNA. ConclusionsMultifarious immunotherapeutic approaches in the context of fully suppressive cART further reduce immune activation, and improve both CD4 T-lymphocyte counts and HIV-1-specific T-cell responses (NCT01130376).

93: Identification of the intracellular location and mechanisms of NOD1-dependent inflammatory responses

Nucleotide oligomerization domain 1 (NOD1) is an intracellular host receptor that senses microbial pathogens by detecting a conserved structure of Gram negative bacterial peptidoglycan (PG). Detection of peptidoglycan by NOD1 ultimately results in a pro-inflammatory cytokine response. However, to date, the intracellular location and the mechanisms whereby NOD1 detects peptidoglycan resulting in the development of pro-inflammatory cytokine responses and autophagy are unknown. We used peptidoglycan-containing bacterial outer membrane vesicles (PG-OMVs) as a tool to elucidate the intracellular location of NOD1 and the mechanisms of NOD1-dependent responses that result in cytokine production and autophagy. Upon entry into host epithelial cells, PG-OMVs from mucosal pathogens induced NOD1-dependent autophagy and IL-8 responses. Fluorescent labelling of peptidoglycan contained within bacterial OMVs revealed that upon entry into host cells, peptidoglycan migrated to early endosomes where it interacted with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response from this location. We showed that migration of PG-OMVs to early endosomes occurred in a NOD1 dependent manner, identifying a previously unknown role for NOD1 in the intracellular migration of peptidoglycan. Most importantly, using fluorescent lifetime imaging microscopy (FLIM)-fluorescence energy transfer (FRET), we were able to show for the first time the direct interaction between bacterial peptidoglycan and NOD1 within host cells. Finally, we found that the NOD1 adaptor protein RIP-2 is essential for the development of NOD1-dependent autophagy and IL-8 production in response to PG-OMVs [1] . This study reveals for the first time the intracellular location and the early recognition events required for the detection of Gram negative bacterial pathogens by NOD1. Moreover, this study is the first to visualise a direct interaction between bacterial peptidoglycan and NOD1. These findings will significantly expand our limited knowledge of the contribution of NOD1 in Gram negative bacterial pathogenesis, innate immunity and inflammatory disorders.

Downregulation of cystathionine β-synthase/hydrogen sulfide contributes to rotenone-induced microglia polarization toward M1 type

Microglia-mediated neuroinflammation is implicated in the pathogenesis of several neurodegenerative disorders. Microglia can be activated and polarized to exert pro- or anti-inflammatory roles in response to specific stimulus. Rotenone is an environmental toxin that has been shown to activate microglia and neuroinflammation. However, the effects and mechanisms of rotenone on microglia polarization are poorly studied. In the present study, we demonstrated that rotenone enhanced the levels of M1 phenotypic genes including TNF-α, iNOS and COX-2/PGE2 but reduced that of M2 markers such as Ym1/2 and IL-10 in mouse primary and immortalized microglia. Moreover, the transcription and protein expression of cystathionine-β-synthase (CBS), as well as hydrogen sulfide (H2S) production were decreased in rotenone-treated primary microglia. Elevating endogenous H2S via CBS over-expression in immortalized microglia not only reduced the expression of pro-inflammatory M1 genes, but also enhanced the anti-inflammatory M2 marker IL-10 production in response to rotenone stimulation as compared to vector-transfected cells. Similarly, pretreatment with H2S donor NaHS (50, 100 and 500μmol/L) attenuated the increases of M1 gene expression triggered by rotenone treatment, and enhanced the M2 gene Ym1/2 expression in mouse primary microglia. In addition, we observed reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine reversed the down-regulation of CBS and H2S generation caused by rotenone in microglia. NaHS pretreatment also decreased the ROS formation in rotenone-stimulated microglia. Taken together, these results reveal that probably via triggering ROS formation, rotenone suppressed the CBS–H2S pathway and thus promoted microglia polarization toward M1 pro-inflammatory phenotype.

Infliximab therapy increases the frequency of circulating CD16(+) monocytes and modifies macrophage cytokine response to bacterial infection.

Crohn's disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)-10] were evaluated at different time-points. To evaluate infliximab-dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16(+) and CD16(-) monocytes and the frequency of TNF(+) cells among CD16(+) and CD16(-) monocyte-derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL-10 macrophage response to bacteria. An infliximab-dependent increase in the frequency of circulating CD16(+) monocytes (particularly the CD14(++) CD16(+) subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16(+) monocytes were higher TNF producers and CD16(+) macrophages from infliximab-treated CD patients showed increased frequency of TNF(+) cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16(+) circulating monocytes, particularly of the CD14(++) CD16(+) subset. Infliximab treatment of CD patients also resulted in increased macrophage IL-10 production in response to bacteria, suggesting an infliximab-induced shift to M2 macrophages.

AIP augments CARMA1-BCL10-MALT1 complex formation to facilitate NF-κB signaling upon T cell activation.

BackgroundThe CARMA1-BCL10-MALT1 (CBM) complex bridges T cell receptor (TCR) signaling to the canonical IκB kinase (IKK)/NF-κB pathway. The CBM complex constitutes a signaling cluster of more than 1 Mio Dalton. Little is known about factors that facilitate the rapid assembly and maintenance of this dynamic higher order complex.FindingsHere, we report the novel interaction of the aryl hydrocarbon receptor (AHR) interacting protein (AIP) and the molecular scaffold protein CARMA1. In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex. Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation.ConclusionsOur data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.

Lactobacillus rhamnosus L34 and Lactobacillus casei L39 suppress Clostridium difficile-induced IL-8 production by colonic epithelial cells.

BackgroundClostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as C. difficile-associated disease (CDAD).With increased severity and failure of treatment in CDAD, new approaches for prevention and treatment, such as the use of probiotics, are needed. Since the pathogenesis of CDAD involves an inflammatory response with a massive influx of neutrophils recruited by interleukin (IL)-8, this study aimed to investigate the probiotic effects of Lactobacillus spp. on the suppression of IL-8 production in response to C. difficile infection.ResultsWe screened Lactobacillus conditioned media from 34 infant fecal isolates for the ability to suppress C. difficile-induced IL-8 production from HT-29 cells. Factors produced by two vancomycin-resistant lactobacilli, L. rhamnosus L34 (LR-L34) and L.casei L39 (LC-L39), suppressed the secretion and transcription of IL-8 without inhibiting C. difficile viability or toxin production. Conditioned media from LR-L34 suppressed the activation of phospho-NF-κB with no effect on phospho-c-Jun. However, LC-L39 conditioned media suppressed the activation of both phospho-NF-κB and phospho-c-Jun. Conditioned media from LR-L34 and LC-L39 also decreased the production of C. difficile-induced GM-CSF in HT-29 cells. Immunomodulatory factors present in the conditioned media of both LR-L34 and LC-L39 are heat-stable up to 100°C and > 100 kDa in size.ConclusionsOur results suggest that L. rhamnosus L34 and L. casei L39 each produce factors capable of modulating inflammation stimulated by C. difficile. These vancomycin-resistant Lactobacillus strains are potential probiotics for treating or preventing CDAD.

Crucial role of the MAPKAP kinases 2 and 3 for pathogen-induced inflammation and their relevance for the immune response of the liver

Crucial role of the MAPKAP kinases 2 and 3 for pathogen-induced inflammation and their relevance for the immune response of the liver

Modulation of antigen-specific T-cells as immune therapy for chronic infectious diseases and cancer.

T-cell responses are induced by antigen presenting cells (APC) and signals from the microenvironment. Antigen persistence and inflammatory microenvironments in chronic infections and cancer can induce a tolerant state in T-cells resulting in hyporesponsiveness, loss of effector function, and weak biochemical signaling patterns in response to antigen stimulation. Although the mechanisms of T-cell tolerance induced in chronic infection and cancer may differ from those involved in tolerance to self-antigen, the impaired proliferation and production of IL-2 in response to antigen stimulation are hallmarks of all tolerant T cells. In this review, we will summarize the evidence that the immune responses change from non-self to “self”-like in chronic infection and cancer, and will provide an overview of strategies for re-balancing the immune response of antigen-specific T cells in chronic infection and cancer without affecting the homeostasis of the immune system.

Cadmium administration affects circulatory mononuclear cells in rats

Although numerous investigations have demonstrated a direct effect of cadmium (Cd) on peripheral blood mononuclear cell (PBMC) activity in humans, there is virtually no data concerning the in vivo impact of this metal on circulatory mononuclear cells. In this study, the effects of a sub-lethal Cd (1 mg/kg) dose were examined in rats 48 h following a single intraperitoneal injection. Cd treatment resulted in increased total peripheral blood leukocyte levels; however, decreases in PBMC numbers were seen. These changes coincided with an accumulation of mononuclear cells in the lungs and an increase in mononuclear cells expressing CD11b. A lack of effect of Cd on spontaneous nitric oxide (NO) production and on iNOS mRNA levels in the PBMC was also noted. Differential effects of Cd on PBMC inflammatory cytokine (IL-1β, TNFα, IL-6, IFNγ, and IL-17) gene expression and production were also seen. Specifically, except for IL-1β (levels increased), there were decreases (relative to controls) in mRNA levels for all the other cytokines examined. While there were no Cd treatment-related changes in spontaneous production of the cytokines assessed, there seemed to be a trend (p = 0.06) toward a decrease in spontaneous IL-6 release. When these harvested cells were stimulated ex vivo, there was no effect from Cd exposure on LPS-stimulated IL-1β and TNFα or on ConA-stimulated IFNγ or IL-17 production, but a decrease in IL-6 production in response to LPS was, again, noted. A preliminary study with a lower Cd dose (0.5 mg/kg) revealed some of the same outcomes noted here (mononuclear cell infiltration into lungs, increases in PBMC IL-1β mRNA levels), but differential (increased IL-17 mRNA levels) or newly detected outcomes (increased levels of IL-1α mRNA) as well. The described effects of the single in vivo exposure to Cd on PBMC might contribute to a better overall understanding of the immunomodulatory potential of this environmental contaminant.

Interactive effects of cerium oxide and diesel exhaust nanoparticles on inducing pulmonary fibrosis

Cerium compounds have been used as a fuel-borne catalyst to lower the generation of diesel exhaust particles (DEPs), but are emitted as cerium oxide nanoparticles (CeO2) along with DEP in the diesel exhaust. The present study investigates the effects of the combined exposure to DEP and CeO2 on the pulmonary system in a rat model. Specific pathogen-free male Sprague–Dawley rats were exposed to CeO2 and/or DEP via a single intratracheal instillation and were sacrificed at various time points post-exposure. This investigation demonstrated that CeO2 induces a sustained inflammatory response, whereas DEP elicits a switch of the pulmonary immune response from Th1 to Th2. Both CeO2 and DEP activated AM and lymphocyte secretion of the proinflammatory cytokines IL-12 and IFN-γ, respectively. However, only DEP enhanced the anti-inflammatory cytokine IL-10 production in response to ex vivo LPS or Concanavalin A challenge that was not affected by the presence of CeO2, suggesting that DEP suppresses host defense capability by inducing the Th2 immunity. The micrographs of lymph nodes show that the particle clumps in DEP+CeO2 were significantly larger than CeO2 or DEP, exhibiting dense clumps continuous throughout the lymph nodes. Morphometric analysis demonstrates that the localization of collagen in the lung tissue after DEP+CeO2 reflects the combination of DEP-exposure plus CeO2-exposure. At 4weeks post-exposure, the histological features demonstrated that CeO2 induced lung phospholipidosis and fibrosis. DEP induced lung granulomas that were not significantly affected by the presence of CeO2 in the combined exposure. Using CeO2 as diesel fuel catalyst may cause health concerns.

Human S100A9 potentiates IL-8 production in response to GM-CSF or fMLP via activation of a different set of transcription factors in neutrophils.

Inflammation is highly regulated by various agents. Unexpectedly, we report here that the damage-associated molecular pattern S100A9 protein, a potent neutrophil activator and inducer of cytokine production in monocytes, is not a direct activator of cytokine production in human neutrophils. However, S100A9 primed IL-8 production in fMLP- and GM-CSF-stimulated neutrophiles via NF-κB and CREB-1, and NF-κB, STAT3 and STAT5, respectively. Pharmacological inhibition confirmed the importance of these transcription factors by significantly decreasing IL-8 production. This is the first time that a different set of transcription factors are shown to be involved in S100A9-primed neutrophils in response to proinflammatory agonist.

Self-education via Ly49 receptor engagement of donor and host iNKT cells following in utero hematopoietic cellular transplantation (TRAN1P.874)

Abstract Invariant NKT (iNKT) cells recognize glycolipid-CD1d complexes via their TCR but also express inhibitory Ly49 receptors (iLy49s) specific for MHC I ligands. However, little is known regarding the importance of iLy49s in the self-recognition of MHC I by iNKT cells during development. We hypothesized that self-engagement of iLy49s during development alters the phenotype and function of self-responsive iNKT cells. We examined this hypothesis in prenatal Balb/c to B6 and B6 to Balb/c allogeneic chimeras that were established by in utero hematopoietic cellular transplantation (IUHCT) of E14 fetal liver light density cells into age-matched fetal recipients. After birth, we compared iLy49 expression and the functional response of iNKT cells in the allogeneic chimeras to age-matched controls. The levels of allospecific iLy49s on host iNKT and donor iNKT were significantly reduced in all organs tested suggesting developmental recognition of both donor and host MHC class I had occurred. In contrast, the expression of irrelevant receptors was not altered. Lastly, iNKT cells from chimeric mice that expressed allospecific iLy49s exhibited significantly increased production of IL-4 in response to TCR stimulation suggesting that a shift toward a regulatory phenotype had occurred. Future studies will directly evaluate the impact of self-recognition by iNKT cells in the developmental pathogenesis of autoimmunity, allergy and allospecific tolerance.

Virus replication and cytokine production in primary alveolar macrophages and airway epithelial cells induced by emerging canine H3N8 influenza virus (LB510)

Canine H3N8 influenza virus was first recognized as a serious infectious pulmonary disease in racing greyhound dogs in 2004. Disease resulted from direct transmission of commonly circulating equine H3N8 virus to dogs.We hypothesized that transmission of equine influenza virus to dogs was characterized by mutations in one or more genes that allowed the virus to replicate more efficiently in dog airway epithelial cells and/or alveolar macrophages and induce higher levels of proinflammatory cytokines. Primary alveolar macrophages and airway epithelial cells were isolated from dogs and inoculated with emergent and endemic canine H3N8 influenza viruses A/canine/Florida /43/2004 (canine/FL/04), canine/FL/05, canine/PA/09 as well as early and more recent equine viruses equine/KY/91, equine/NY/99 and equine/OH/03.Cytokines responses were measured by multiplex protein assay and/or quantitative PCR. Emergent and endemic canine viruses replicated to higher titers in dog macrophages than did the equine viruses from 1999 and 2003. Only endemic canine viruses from 2005 and 2009 replicated to a higher titer in isolated dog airway epithelial cells than did equine viruses from 1999 and 2005. Induction of TNF‐alpha in macrophages by equine and canine viruses was highly variable among macrophage isolates from different dogs. No significant differences were measured in epithelial cell production of IL‐8 in response to canine or equine H3N8 virus inoculation. Although small differences in productive virus replication were detected for canine influenza virus isolates compared to equine influenza virus isolates in macrophages and epithelial cells, differences in cytokine production were not detected.Grant Funding Source: Supported by NIH AI101625