Biomechanical forces enhance directed migration and activation of bone marrow-derived dendritic cells.

Mechanical forces are pervasive in the inflammatory site where dendritic cells (DCs) are activated to migrate into draining lymph nodes. For example, fluid shear stress modulates the movement patterns of DCs, including directness and forward migration indices (FMIs), without chemokine effects. However, little is known about the effects of biomechanical forces on the activation of DCs. Accordingly, here we fabricated a microfluidics system to assess how biomechanical forces affect the migration and activity of DCs during inflammation. Based on the structure of edema, we proposed and experimentally analyzed a novel concept for a microchip model that mimicked such vascular architecture. The intensity of shear stress generated in our engineered chip was found as 0.2-0.6 dyne/cm2 by computational simulation; this value corresponded to inflammation in tissues. In this platform, the directness and FMIs of DCs were significantly increased, whereas the migration velocity of DCs was not altered by shear stress, indicating that mechanical stimuli influenced DC migration. Moreover, DCs with shear stress showed increased expression of the DC activation markers MHC class I and CD86 compared with DCs under static conditions. Taken together, these data suggest that the biomechanical forces are important to regulate the migration and activity of DCs.

Extracellular Vesicle Surface Signatures in IPF Patients: A Multiplex Bead-Based Flow Cytometry Approach.

Background: Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods: Ninety IPF patients (median age and interquartile range (IQR) 71 (66-75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, (Miltenyi Biotec, Bergisch-Gladbach, Germany), to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls (p = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively). CD42a was lower in IPF subjects than in controls (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls (p = 0.0007, p = 0.0050, p = 0.0139, and p = 0.0335, respectively). Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate (p = 0.0243, hazard ratio: 0.52 (95%CI 0.29-0.92); the same was true for CD8 (cut-off 1.53, p = 0.0309, hazard ratio: 1.39 (95%CI 0.75-2.53). Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.

Scrib and Dlg1 polarity proteins regulate Ag presentation in human dendritic cells.

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

Increases of CD80 and CD86 Expression on Peripheral Blood Cells and their Gene Polymorphisms in Autoimmune Thyroid Disease.

The prognosis of autoimmune thyroid diseases (AITDs), such as Graves' disease (GD) and Hashimoto's disease (HD), are difficult to predict. Both CD80 and CD86 costimulatory signals promote T cell activation in cooperation with T cell receptor signal. To clarify whether any association between CD80 and CD86 and the pathogenesis of AITD exist, we examined the expressions and gene polymorphisms of CD80 and CD86. We examined the expressions of CD80 and CD86 proteins on peripheral blood cells by flowcytometry and genotyped CD80 and CD86 gene polymorphisms by PCR-RFLP and Taqman PCR methods. In the analysis of the Blymphocytes elevated CD80+ cells (>8%) were found more often in the patients than in control subjects, and also it was more frequent in patients with intractable GD than in those with GD in remission (p= .0176). The mean fluorescence intensity of CD86 expression on monocytes was higher in GD and HD patients than in control subjects (p= <0.0001 and p= .0017, respectively). CD80 rs1599795 T allele carriers were more frequent in patients with severe HD than in those with mild HD. CD86 rs2715267 AA genotype was more frequent in HD patients than in controls. In conclusion, the expressions of CD80 on Bcells and of CD86 on monocytes were increased in peripheral blood from patients with AITD, especially in severe cases, and their gene polymorphisms are associated with the susceptibility and the severity of HD.

Rosiglitazone polarizes microglia and protects against pilocarpine-induced status epilepticus.

AIMS: Activated microglia have been found in the forebrains and hippocampi of temporal lobe epilepsy (TLE) patients and status epileptic (SE) animal models. The peroxisome proliferator-activated receptor γ (PPAR γ) agonist rosiglitazone has been shown to prevent microglial activation. However, its role in pilocarpine-induced status epilepticus remains unknown. We aimed to examine the effect of the PPAR γ agonist rosiglitazone in protecting against pilocarpine-induced status epileptic resulting from over-activation and to explore phenotypic changes in microglia as the underlying mechanism. METHODS: Male C57BL/6 mice were assigned to three groups: the control group, pilocarpine-induced (SE) group, and rosiglitazone-treated (SE+Rosi) group. Status epileptic mice were administered 300 mg/kg pilocarpine via intraperitoneal injection. SE+Rosi mice were administered rosiglitazone (0.1 mg/kg, i.p.) after SE. Flow cytometry, immunofluorescence staining, and quantitative real-time PCR were used to examine the activation of and phenotypic changes in microglia in the brain and to evaluate neuroinflammation. RESULTS: We found that the expression of proinflammatory CD86 and iNOS was increased and that the expression of antiinflammatory CD206 and Arg-1 was decreased in the brains of pilocarpine-induced SE mice compared to control mice. The mRNA levels of proinflammatory and antiinflammatory cytokines were not significantly changed in the brain. Rosiglitazone treatment significantly inhibited the proinflammatory polarization of microglia and rescued neuron loss in the temporal lobe and hippocampi of the brain after SE. CONCLUSION: Rosiglitazone reverses microglial polarization in the brains of SE mice and also affords neuroprotection against pilocarpine-induced status epilepticus without inducing significant changes in brain inflammation.

The HaCaT/THP-1 Cocultured Activation Test (COCAT) for skin sensitization: a study of intra-lab reproducibility and predictivity.

The Cocultured Activation Test (COCAT) consists of cocultured HaCaT (human keratinocyte cell line) and THP-1 cells (surrogate of antigen presenting cells). Individually, these cell lines are used to address key event 2 and 3 of the skin sensitization Adverse Outcome Pathway (AOP). Their exposure in coculture was found to have the potential to increase their response to sensitizing chemicals, enable the detection of pro-haptens and support the identification of skin sensitization potency. The present study was undertaken to assess the predictive capacity of COCAT to both skin sensitization hazard and potency and to assess the intra-laboratory reproducibility of COCAT based on the blind testing of chemicals. Results showed a reproducibility between runs of 80 % for 15 coded chemicals. 100 % sensitivity (9/9), 75 % specificity (3/4) and 92.3 % accuracy (12/13) was found for skin sensitization hazard prediction, while the tests of two chemicals were inconclusive. Including additional chemicals tested during the optimization phase in addition to the blind tested chemicals, the skin sensitization UN GHS sub-categories were correctly predicted for 85.7 % (12/14) Sub-category 1A chemicals, 83.3 % (10/12) Sub-category 1B chemicals and 92.3 % (12/13) 'No Category' chemicals, resulting in an overall accuracy of 87.4 % (34/39). The present study shows the COCAT to be a promising method for the identification of skin sensitization hazard and potency sub-categorization according to the UN GHS classification.

Differential expression of CD73, CD86 and CD304 in normal vs. leukemic B-cell precursors and their utility as stable minimal residual disease markers in childhood B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. METHODS: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. RESULTS: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. CONCLUSIONS: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells.

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.

NP30 stimulates Th17 differentiation through DC in Schistosomiasis Japonicum.

The murine monoclonal anti-idiotypic antibody, NP30, is a potential vaccine candidate against Schistosoma japonicum. Previous studies have revealed that NP30 has an immunoregulatory effect, but the underlying mechanism for this effect remains unknown. This study shows that NP30 induces dendritic cell (DC) maturation and increases the production of pro-inflammatory cytokines. The expression of CD86 and MHC II was upregulated in DCs following stimulation with NP30 in vitro. Moreover, NP30 induced Th17 polarization by increasing the production of IL-6 and TGF-ß. In vivo, Th17 differentiation was induced by the production of key pro-inflammatory cytokines, including IL-6and TGF-ß, from DCs of NP30-immunized mice. These results indicate that NP30 promotes Th17 polarization through DC activation, preventing serious schistosomiasis.

Immune modulation by silencing CD80 and CD86 production in dendritic cells using small hairpin RNA to reduce heart transplant rejection.

RNA interference (RNAi) plays a potential role in organ transplantation. Small hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence the expression of a target gene. We constructed shRNA targeting on the cluster of differentiation 80 (CD80, B7-1) and the cluster of differentiation 86 (CD86, B7-2) and transfected it into dendritic cells (DCs). Fluorescence real-time PCR and flow cytometry confirmed the gene-silencing effect. Interleukin-2 (IL-2) mRNA expression level decreased in T cells that were cocultured with pB7-shRNA-transfected DCs. For in-vivo experiment, we built mice models of abdominal heterotopic heart transplantation and transfused the models with pB7-shRNA-transfected donor-derived DCs. The survival time of the transplanted heart increased; the grade of organ rejection decreased. IL-2 mRNA expression level decreased and it was positively correlated with the grade of organ rejection.

Leptin is essential for microglial activation and neuropathic pain after preganglionic cervical root avulsion.

AIMS: Preganglionic cervical root avulsion (PCRA) affects both the peripheral and central nervous systems and is often associated with neuropathic pain. Unlike peripheral nerve injuries (PNI), central lesions caused by disruption of cervical roots from the spinal cord following PCRA contribute to the generation of neuropathic pain. Leptin is involved in the development of neuropathic pain after PNI by affecting neurons. However, whether leptin is involved in microglial activation leading to neuropathic pain after PCRA is unknown. MAIN METHODS: Preganglionic avulsion of the left 6th-8th cervical roots was performed in C57B/6J mice and leptin-deficient mice. A leptin antagonist or leptin was administered to C57B/6J mice and leptin-deficient mice after injury, respectively. The expression pattern of spinal and supraspinal microglia was examined by immunofluorescent staining. Von Frey filaments were used to test pain sensitivity. KEY FINDINGS: Leptin is essential for the development of neuropathic pain after PCRA. Allodynia was absent in the leptin-deficient mice and the mice administered the leptin antagonist. We also found that leptin deficiency or the administration of its antagonist inhibited the development of microgliosis in the dorsal horn and brainstem. Furthermore, increase in the expression of CD86 and iNOS, and Wallerian degeneration were noted in the spinal cord. The administration of exogenous leptin to leptin-deficient mice reversed these effects. SIGNIFICANCE: We concluded that leptin is involved in the proliferation and activation of microglia, which in turn enhances the development of neuropathic pain. Blocking the effects of leptin might be a target for the treatment of neuropathic pain after PCRA.

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

Capparis spinosa Fruit Ethanol Extracts Exert Different Effects on the Maturation of Dendritic Cells.

Capparis spinosa L. (C. spinosa) has been used as food and traditional medicine and shows anti-inflammatory and anti-oxidant activities. Here, we prepared the C. spinosa fruit ethanol extracts (CSEs) using different procedures and investigated the effects of CSE on the maturation of mouse bone marrow-derived dendritic cells (DCs) in the absence or presence of lipopolysaccharide (LPS). DC maturation and cytokine production were detected by flow cytometry and ELISA, respectively. We obtained three different CSEs and dissolved in water or DMSO, named CSE2W, CSEMW, CSE3W, CSE2D, CSEMD, and CSE3D, respectively. These CSEs showed different effects on DC maturation. CSEMW and CSEMD significantly increased the expressions of CD40, CD80, and CD86, in a dose-dependent manner. CSE2W and CSE2D also showed a modest effect on DC maturation, which enhanced the expression of CD40. CSE3W and CSE3D did not change DC maturation but suppressed LPS-induced DC maturation characterized by the decreased levels of CD40 and CD80. CSE3W and CSE3D also significantly inhibited the secretions of IL-12p40, IL-6, IL-1ß, and TNF-α induced by LPS. CSE3W further increased the level of IL-10 induced by LPS. Moreover, CSE3D suppressed LPS-induced DC maturation in vivo, which decreased the expressions of CD40 and CD80. These results suggested that CSE3W and CSE3D might be used to treat inflammatory diseases.

Modification of host dendritic cells by microchimerism-derived extracellular vesicles generates split tolerance.

Maternal microchimerism (MMc) has been associated with development of allospecific transplant tolerance, antitumor immunity, and cross-generational reproductive fitness, but its mode of action is unknown. We found in a murine model that MMc caused exposure to the noninherited maternal antigens in all offspring, but in some, MMc magnitude was enough to cause membrane alloantigen acquisition (mAAQ; "cross-dressing") of host dendritic cells (DCs). Extracellular vesicle (EV)-enriched serum fractions from mAAQ+, but not from non-mAAQ, mice reproduced the DC cross-dressing phenomenon in vitro. In vivo, mAAQ was associated with increased expression of immune modulators PD-L1 (programmed death-ligand 1) and CD86 by myeloid DCs (mDCs) and decreased presentation of allopeptide+self-MHC complexes, along with increased PD-L1, on plasmacytoid DCs (pDCs). Remarkably, both serum EV-enriched fractions and membrane microdomains containing the acquired MHC alloantigens included CD86, but completely excluded PD-L1. In contrast, EV-enriched fractions and microdomains containing allopeptide+self-MHC did not exclude PD-L1. Adoptive transfer of allospecific transgenic CD4 T cells revealed a "split tolerance" status in mAAQ+ mice: T cells recognizing intact acquired MHC alloantigens proliferated, whereas those responding to allopeptide+self-MHC did not. Using isolated pDCs and mDCs for in vitro culture with allopeptide+self-MHC-specific CD4 T cells, we could replicate their normal activation in non-mAAQ mice, and PD-L1-dependent anergy in mAAQ+ hosts. We propose that EVs provide a physiologic link between microchimerism and split tolerance, with implications for tumor immunity, transplantation, autoimmunity, and reproductive success.

Curcumin reduces lung inflammation via Wnt/ß-catenin signaling in mouse model of asthma.

OBJECTIVES: Asthma is a chronic inflammatory, heterogeneous airway disease affecting millions of people around the world. Curcumin has been found to have anti-inflammatory and antifibrosis effects. Researchers reported that curcumin regulated Wnt/ß-catenin signaling in lots of cells. However, whether curcumin regulates the levels of Wnt/ß-Catenin signaling in lung tissues and DCs (dendritic cells) remains unclear. In this study, we assessed the effects of curcumin on DCs and asthma. METHODS: C57BL/6 mice immunized with OVA (ovalbumin) were challenged thrice with an aerosol of OVA every second day for 8 days. Dexamethasone or curcumin was administered intraperitoneally to OVA-immunized C57BL/6 mice on day 24 once a day for 9 days. Mice were analyzed for effects of curcumin on asthma, inflammatory cell infiltration and cytokine levels in lung tissue. DCs were isolated from mouse bone morrow. The surface markers CD40, CD86 and CD11c of DCs was detected by FACS (fluorescence activated cell sorting) and the function of DCs was detected by mixed lymphocyte reaction. The expression of GSK-3ß and ß-catenin was detected by Western Blot. RESULTS: Results showed that OVA increased the number of inflammatory factors in BALF (bronchoalveolar lavage fluid), elevated lung inflammation scores in mice. Curcumin dose-dependently reversed the alterations induced by OVA in the asthmatic mice. Curcumin activated Wnt/ß-catenin signaling pathway in DCs and asthmatic mouse lungs. CONCLUSIONS: Curcumin could influence the morphology and function of DCs, ease asthma symptom and inflammatory reaction through the activation of Wnt/ß-catenin signaling. These results provide new evidence new evidence for application of curcumin on asthma.

Altered immune response of immature dendritic cells following dengue virus infection in the presence of specific antibodies.

Dengue virus (DENV) replication is known to prevent maturation of infected dendritic cells (DCs) thereby impeding the development of adequate immunity. During secondary DENV infection, dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), however how dengue-antibody complexes (DENV-IC) influence the phenotype of DCs remains elusive. Here, we evaluated the maturation state and cytokine profile of immDCs exposed to DENV-ICs. Indeed, DENV infection of immDCs in the absence of antibodies was hallmarked by blunted upregulation of CD83, CD86 and the major histocompatibility complex molecule HLA-DR. In contrast, DENV infection in the presence of neutralizing antibodies triggered full DC maturation and induced a balanced inflammatory cytokine response. Moreover, DENV infection under non-neutralizing conditions prompted upregulation of CD83 and CD86 but not HLA-DR, and triggered production of pro-inflammatory cytokines. The effect of DENV-IC was found to be dependent on the engagement of FcγRIIa. Altogether, our data show that the presence of DENV-IC alters the phenotype and cytokine profile of DCs.

Dendritic Cells Coordinate the Development and Homeostasis of Organ-Specific Regulatory T Cells.

Although antigen recognition mediated by the T cell receptor (TCR) influences many facets of Foxp3(+) regulatory T (Treg) cell biology, including development and function, the cell types that present antigen to Treg cells in vivo remain largely undefined. By tracking a clonal population of Aire-dependent, prostate-specific Treg cells in mice, we demonstrated an essential role for dendritic cells (DCs) in regulating organ-specific Treg cell biology. We have shown that the thymic development of prostate-specific Treg cells required antigen presentation by DCs. Moreover, Batf3-dependent CD8α(+) DCs were dispensable for the development of this clonotype and had negligible impact on the polyclonal Treg cell repertoire. In the periphery, CCR7-dependent migratory DCs coordinated the activation of organ-specific Treg cells in the prostate-draining lymph nodes. Our results demonstrate that the development and peripheral regulation of organ-specific Treg cells are dependent on antigen presentation by DCs, implicating DCs as key mediators of organ-specific immune tolerance.

MicroRNA-487b Is a Negative Regulator of Macrophage Activation by Targeting IL-33 Production.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate a broad spectrum of biological processes, including immune responses. Although the contributions of miRNAs to the function of immune cells are beginning to emerge, their specific roles remain largely unknown. IL-33 plays an important role in macrophage activation for innate host defense and proinflammatory responses. In this study, we report that miR-487b can suppress the levels of mRNA and protein for IL-33 during the differentiation of bone marrow-derived macrophages (BMDMs). This results in inhibition of IL-33-induced expression of Ag-presenting and costimulatory molecules and proinflammatory mediators. A luciferase assay showed that miR-487b binds to the IL-33 3'-untranslated region. We also confirmed that IL-33 directly promotes the activation of BMDMs by increasing the expression of MHC class I, MHC class II, CD80/CD86, and inducible NO synthase (iNOS) in a dose-dependent manner. Exposure of BMDMs to the TLR4 ligand, LPS, decreased miR-487b expression, increased IL-33 transcript levels, and induced the production of proinflammatory mediators (e.g., iNOS, IL-1ß, IL-6, and TNF-α). Treatment with a specific inhibitor of miR-487b function also resulted in increased levels of IL-33 mRNA, which augmented LPS-induced expression of these inflammatory mediators in macrophages. Collectively, our results indicate that miR-487b plays a negative regulatory role in macrophages by controlling the levels of IL-33 transcript and protein to fine-tune innate immune host defense and proinflammatory responses of these cells. Thus, miR-487b plays an important role in the regulation of macrophage homeostasis and activation by targeting IL-33 transcripts.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells.

Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1ß or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.