MiR-146a modulates macrophage polarization in systemic juvenile idiopathic arthritis by targeting INHBA

Monocytes from patients with systemic juvenile idiopathic arthritis (SJIA) have both features of classical activated M1 and alternatively activated M2 macrophages. An increasing number of studies have indicated that microRNAs (miRNAs) are critical regulators of monocyte polarization. Here, we focused on miR-146a expression in SJIA and investigated the function of miR-146a in monocyte polarization. We found that miR-146a expression was highly up-regulated in SJIA monocytes and correlated with the systemic features. miR-146a was expressed at a higher level in monocytes polarized with M2 conditions than those polarized with M1 conditions. miR-146a overexpression significantly decreased the production of M1 phenotype markers such as IL-6, IL-12, TNF-α, CD86 and iNOS in M1 macrophages, but increased the production of M2 marker genes such as Arg1, CCL17, CCL22 and CD206 in M2 macrophages. Conversely, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. We subsequently demonstrated that miR-146a targeted the 3′-untranslated region (UTR) of INHBA to inhibit its expression. Additionally, INHBA overexpression rescued the reduced IL-6, IL-12, and TNF-α levels induced by miR-146a overexpression in M1 macrophages, and rescued the increased Arg1, CCL17, and CCL22 levels induced by miR-146a overexpression in M2 macrophages. Similarly, the effects of miR-146a inhibition in monocyte polarization were all partly reversed by INHBA inhibition. Taken together, the data suggest that miR-146a serves as a molecular regulator in monocyte polarization and might play an important role in monocytes from patients with SJIA.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.     

Enhanced and persistent levels of interleukin (IL)‐17+CD4+ T cells and serum IL‐17 in patients with early inflammatory arthritis

Prognosis of patients with early inflammatory arthritis (EIA) is highly variable. The aim of this study was to compare, longitudinally and cross-sectionally, the levels of cytokine-expressing cells in peripheral blood (PB) from patients with EIA to those in established rheumatoid arthritis (RA) and healthy controls (HC). PB mononuclear cells from HC (n = 30), patients with EIA (n = 20) or RA (n = 38) were stimulated with phorbol myristate acetate (PMA)/ionomycin for 3 h, and stained for cell markers and cytokines. Serum cytokines and chemokines were measured by Luminex. Patients with EIA were reassessed at 6 and 12 months. The percentage of interleukin (IL)-17⁺interferon (IFN)-γ⁻CD4⁺ T cells [T helper type 17 (Th17)] was increased in RA and EIA versus HC. Serum IL-1β, IL-2, IL-4 IL-17 and macrophage inflammatory protein (MIP)-1α were increased in RA and EIA versus HC. IL-1Ra, IL-15 and IFN-α were increased in EIA versus HC. IL-6 and tumour necrosis factor (TNF)-α was increased in RA but not EIA versus HC. Disease activity scores in EIA patients improved over 12 months'' treatment. Th17 percentage at baseline was correlated with both rheumatoid factor (RF) titre and functional deficit at 12 months. Baseline levels of serum granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-6 and IL-8 were correlated with Larsen score at 12 months. There were no significant changes in cytokine-expressing CD4⁺T cells over time, although the percentage of IL-6⁺ monocytes increased. IL-17⁺CD4⁺ T cells and serum IL-17 levels are increased in EIA. IL-6-expressing monocytes increase during the first year of disease, irrespective of disease-modifying anti-rheumatic drug (DMARD) therapy. We observed incomplete clinical responses, suggesting EIA patients need more intensive early therapy.     

Schistosoma japonicum infection induces macrophage polarization

The role of macrophages （MФ） as the first line of host defense is well accepted. These cells play a central role in orchestrating crucial functions during schistosomal infection. Thus, understanding the functional diversity of these cells in the process of infection as well as the mechanisms underlying these events is crucial for developing disease control strategies. In this study, we adopted a Mqb polarization recognition system. M1 macrophage was characterized by expressing CD16/32, IL-12 and iNOS. M2 macrophage was characterized by expressing CD206, IL-10 and arg-1. In vivo （mouse peritoneal macrophages of different infection stages were obtained） and in vitro （different S. japonicum antigens were used to stimulate RAW264.7） were characterized by using the above mentioned system. NCA and ACA stimulated RAW264.7 express significantly higher levels of IL-12 while significantly higher levels of IL-10 were detected after soluble egg antigen （SEA） stimulation. The results showed that dramatic changes of antigen in the microenvironment before and after egg production led to macrophage polarization. Furthermore, through TLR blocking experiments, the TLR4 signaling pathway was found to play a role in the process of macrophage polarization toward M1. Our data suggest that macrophage polarization during S. japonicum infection had significant effects on host immune responses to S. japonicum.     

Porphyromonas gingivalis Lipopolysaccharide Weakly Activates M1 and M2 Polarized Mouse Macrophages but Induces Inflammatory Cytokines

Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth''s supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 μg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages.     

Interleukin‐7 supports survival of T‐cell receptor‐β‐expressing CD4− CD8− double‐negative thymocytes

Among the milestones that occur during T-cell development in the thymus is the expression of T-cell receptor-β (TCR-β) and the formation of the pre-TCR complex. Signals emanating from the pre-TCR trigger survival, proliferation and differentiation of T-cell precursors. Although the pre-TCR is essential for these cell outcomes, other receptors, such as Notch and CXCR4, also contribute. Whether interleukin-7 (IL-7) participates in promoting the survival or proliferation of pre-TCR-expressing cells is controversial. We used in vitro and in vivo models of T-cell development to examine the function of IL-7 in TCR-β-expressing thymocytes. Culturing TCR-β-expressing CD4⁻ CD8⁻ double-negative thymocytes in an in vitro model of T-cell development revealed that IL-7 reduced the frequency of CD4⁺ CD8⁺ double-positive thymocytes at the time of harvest. The mechanism for this change in the percentage of double-positive cells was that IL-7 promoted the survival of thymocytes that had not yet differentiated. By preserving the double-negative population, IL-7 reduced the frequency of double-positive thymocytes. Interleukin-7 was not required for proliferation in the in vitro system. To follow this observation, we examined mice lacking CD127 (IL-7Rα). In addition to the known effect of CD127 deficiency on T-cell development before TCR-β expression, CD127 deficiency also impaired the development of TCR-β-expressing double-negative thymocytes. Specifically, we found that Bcl-2 expression and cell cycle progression were reduced in TCR-β-expressing double-negative thymocytes in mice lacking CD127. We conclude that IL-7 continues to function after TCR-β is expressed by promoting the survival of TCR-β-expressing double-negative thymocytes.     

Evolution of a CD3+/CD+ α/ β T-Cell Receptor+ Mature T-Cell Clone from CD3-CD7+ Sorted Human Bone Marrow Cells

In order to study extrathymic differentiation in vitro, CD7⁺CD3^- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCRδ1 (TCRγ/δ-specific) or WT31 (TCR2, α/β-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3⁺, CD4⁺, CD8^-, TCR2⁺, TCR1^-, and was further characterized as CD2⁺, CD45RO⁺, CD16^-, and CD56^-. The presence of mRNA for TCR α and γ but not ,and γ chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted “natural killer (NK)-like” lysis of K562 target cells, with no autocytotoxicity detected. Tle NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimulation, the cells secreted tumor necrosis factor α and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-δ, and GM-CSF in these cells after stimulation with PHA and B-LCL.These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.     

Mice genetically inactivated in interleukin‐17A receptor are defective in long‐term control of Mycobacterium tuberculosis infection

Interleukin-17A (IL-17A), a pro-inflammatory cytokine acting on neutrophil recruitment, is known to play an important role during Mycobacterium tuberculosis infection, but the role of IL-17A receptor signalling in immune defence against this intracellular pathogen remains poorly documented. Here we have analysed this signalling using C57BL/6 mice genetically inactivated in the IL-17 receptor A subunit (IL-17RA^−/−). Although early after infection bacterial growth was controlled to the same extent as in wild-type mice, IL-17RA^−/− mice were defective in exerting long-term control of M. tuberculosis infection, as demonstrated by a progressively increasing pulmonary bacterial burden and shortened survival time. Compared with infected wild-type mice, IL-17RA^−/− mice showed impaired recruitment of neutrophils to the lungs at the early but not the late stage of infection. Pulmonary tumour necrosis factor-α, IL-6 and particularly IL-10 levels were decreased in the absence of IL-17RA signalling, whereas IL-1β was increased. CD4⁺-mediated and γδ-mediated IL-17A production was dramatically increased in IL-17RA^−/− mice (confirming part of their phenotype), whereas production of interferon-γ and expression of the bactericidal enzyme inducible nitric oxide synthase were not affected. Collectively, our data suggest that early but not late neutrophil recruitment is essential for IL-17A-mediated long-term control of M. tuberculosis infection and that a functional interferon-γ response is not sufficient to control M. tuberculosis growth when the IL-17RA pathway is deficient. As treatment of auto-immune diseases with anti-IL-17A antibodies is actually being tested in clinical studies, our data suggest that caution should be taken with respect to possible reactivation of tuberculosis.     

The chemokine CCL18 causes maturation of cultured monocytes to macrophages in the M2 spectrum

The observation that human monocytes cultured in the presence of the chemokine CCL18 showed increased survival, led us to profile cytokine expression in CCL18-stimulated versus control cultures. CCL18 caused significantly increased expression of chemokines (CXCL8, CCL2, CCL3 and CCL22), interleukin-10 (IL-10) and platelet-derived growth factor, but no up-regulation of M1 cytokines IL-1β or IL-12. CCL18-stimulated monocytes matured into cells with morphological resemblance to IL-4-stimulated macrophages, and expressed the monocyte marker CD14 as well the M2 macrophage markers CD206 and 15-lipoxygenase, but no mature dendritic cell markers (CD80, CD83 or CD86). Functionally, CCL18-stimulated macrophages showed a high capacity for unspecific phagocytosis and for pinocytosis, which was not associated with an oxidative burst. These findings suggest that CCL18-activated macrophages stand at the cross-roads between inflammation and its resolution. The chemokines that are produced in response to CCL18 are angiogenic and attract various leucocyte populations, which sustain inflammation. However, the capacity of these cells to remove cellular debris without causing oxidative damage and the production of the anti-inflammatory IL-10 will initiate termination of the inflammatory response. In summary, CCL18 induces an M2 spectrum macrophage phenotype in the absence of IL-4.     

小鼠ANA-1 巨噬细胞不同极化状态下补体组分mRNA 动态分析

目的：观察小鼠ANA-1巨噬细胞不同极化状态下补体相关组分的mRNA动态变化。方法：〖HTSS〗分别以LPS（1 μg/ml）、IL-4（20 ng/ml）体外刺激小鼠ANA-1细胞,于刺激后3、8、12及24 h时间点收获细胞,Trizol法提取细胞总RNA,采用实时荧光定量PCR法检测IL-1β、CCL2、Arg-1的表达,判断细胞极化情况,检测胞内C3、CfB、CRIg、C1q补体分子mRNA的动态表达。结果：细胞极化情况：刺激3 h后LPS组IL、1β、CCL2 的mRNA表达上调,12 h达高峰（2 -△△Ct 分别为297.0±31.0和19.9±3.3）;在IL-4组中以Arg-1mRNA表达上调显著,于24 h达高峰（2 -△△Ct ：27.3±9.1）,提示LPS组细胞向M1方向极化,IL-4组向M2方向极化。细胞内补体mRNA的动态：两极化组的相应补体组分均表现为不同程度的上调,其中：C1q、C3在M2极化组刺激8 h后显著上调,刺激12 h时达高峰（2 -△△Ct 分别为94.9±12.9和11.3±2.4）;CfB因子在M1极化组中显著上调,以刺激12 h表达上调达高峰（2 -△△Ct 为61.4±6.2）;CRIg在两组中均在24 h时表达上调且具有显著性差异（ P <0.05）。结论： LPS/IL-4刺激3 h后,ANA-1细胞分别向M1、M2方向极化;不同补体组分mRNA的表达在两组中均上调但表达谱不同,其中C1q、C3和CRIg在M2极化组中上调更为显著,而CfB因子在M1极化组中显著上调;除CRIg外,C1q、C3及CfB因子均在刺激12 h时上调达高峰,上述结果提示补体组分的变化可能与极化的巨噬细胞功能有关。     

免疫补体调节蛋白C1 抑制物对巨噬细胞极化的作用

目的:研究免疫补体调节蛋白C1 抑制物(C1INH)调节巨噬细胞向经典活化巨噬细胞(M1)和选择性激活巨噬细胞(M2)的极化作用。方法:首先从人血中分离单核细胞后,单核细胞在巨噬细胞集落刺激因子(M-CSF)或粒细胞-巨噬细胞集落刺激因子(GM-CSF)作用下转变为静态巨噬细胞;静态巨噬细胞在干扰素-γ(IFN-γ)或白细胞介素4(IL-4)+IL-13 刺激作用下分别转化为M1 或M2。通过流式细胞仪观察C1INH 对巨噬细胞CD14、CD163、CD206 表型标志物的作用;应用RT-PCR 分析C1INH 对巨噬细胞细胞因子、趋化因子、相关酶基因表达影响;利用Western blot 方法,探讨在炎症条件下C1INH 对巨噬细胞CD14 结合Toll 样受体4(TLR4)的影响。结果:C1INH 抑制M-CSF 源性M1 的CD14、CD163 和GM-CSF 源性M2 的CD206 表型。C1INH 减少M1 的肿瘤坏死因子(TNF-α)和IL-6 表达,而增加M2 的15 脂氧合酶(ALOX15)和IL-10 表达。C1INH 抑制M1 的诱导型一氧化氮合酶(iNOS)mRNA,而提高M2 的精氨酸酶1(Arg1)mRNA 表达。C1INH 促进M1 的杀菌活性和M2 对菌的吞噬功能。C1INH 阻断CD14-TLR4 介导信号传导通路。结论:C1INH 调节巨噬细胞极化。     

Heme Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release

The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-α and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNγ) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163⁺ tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.     

Effect of rTsP53 on the M1/M2 activation of bone-marrow derived macrophage in vitro

We investigated that if rTsP53 could be used to activate bone-marrow derived macrophage (BMDM) into M2 macrophage and stop M1 macrophage activation. After 72 h incubation in blank culture medium, cells with PE-CCR7 (-) and FITC-CD206 (-) was extracted and its mean proportion was 92.30 ± 0.22%. With the stimulation of 20 μg/ml IFN-γ for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 16.24 ± 0.82%. With the stimulation of IL-3/IL-14 (both 10 μg/ml) for 72 h, cells with FICT-CD206 (+) was extracted and its mean proportion was 87.32 ± 4.29%. Co-incubation with different dose of rTsP53 (0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 10 μg/ml, respectively) for 72 h, FITC-CD206 (+) macrophage was extracted. The mean proportion in each group was 1.09 ± 0.22%, 2.13 ± 0.13%, 4.91 ± 0.07%, 5.48 ± 0.29%, 9.81 ± 0.06%, 12.83 ± 0.55%, 17.87 ± 0.02%, respectively. The dose of rTsP53 was significantly positive correlated to the proportion of FITC-CD206 (+) macrophage. Co-incubation with 20 μg/ml IFN-γ and 5 μg/ml rTsP53 for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 10.60 ± 0.19%. Compared to that of mere co-incubation with IFN-γ, there was significant difference between the two groups. ELISA showed that Th1 cytokines’ (IFN-γ, IL-6 and TNF-α) level decreased in the culture medium supernatant of BMDM co-incubated with rTsP53. There was negative correlation between the Th1 cytokines’ level and the dose of rTsP53. Both Th2 cytokines (IL-4 and IL-13) and regulatory cytokines in the culture medium increased. There was positive correlation between the Th2 cytokines’ level and the dose of rTsP53. There was also positive correlation between the regulatory cytokines’ level and the dose of rTsP53. Compared to that of BMDM co-incubated with IFN-γ, levels of TNF-α and IL-6 were significant lower than that of BMDM co-incubated with both IFN-γ and rTsP53 (both P < 0.05), while the levels of IL-4 and TGF-β were significant higher (both P < 0.05). There was no significant difference in the levels of IL-13 and IL-10 between the two groups.     

Distribution and prognostic impact of microglia/macrophage subpopulations in gliomas

While the central nervous system is considered an immunoprivileged site and brain tumors display immunosuppressive features, both innate and adaptive immune responses affect glioblastoma (GBM) growth and treatment resistance. However, the impact of the major immune cell population in gliomas, represented by glioma‐associated microglia/macrophages (GAMs), on patients’ clinical course is still unclear. Thus, we aimed at assessing the immunohistochemical expression of selected microglia and macrophage markers in 344 gliomas (including gliomas from WHO grade I–IV). Furthermore, we analyzed a cohort of 241 IDH1R132H‐non‐mutant GBM patients for association of GAM subtypes and patient overall survival. Phenotypical properties of GAMs, isolated from high‐grade astrocytomas by CD11b‐based magnetic cell sorting, were analyzed by immunocytochemistry, mRNA microarray, qRT‐PCR and bioinformatic analyses. A higher amount of CD68‐, CD163‐ and CD206‐positive GAMs in the vital tumor core was associated with beneficial patient survival. The mRNA expression profile of GAMs displayed an upregulation of factors that are considered as pro‐inflammatory M1 (eg, CCL2, CCL3L3, CCL4, PTGS2) and anti‐inflammatory M2 polarization markers (eg, MRC1, LGMN, CD163, IL10, MSR1), the latter rather being associated with phagocytic functions in the GBM microenvironment. In summary, we present evidence that human GBMs contain mixed M1/M2‐like polarized GAMs and that the levels of different GAM subpopulations in the tumor core are positively associated with overall survival of patients with IDH1R132H‐non‐mutant GBMs.     

Effects of CXCL10 on dendritic cell and CD4+ T-cell functions during Leishmania amazonensis infection

Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously reported that treatment with CXCL10 activates macrophage (MΦ) effector function(s) in parasite killing and significantly delays lesion development in susceptible C57BL/6 mice via enhanced gamma interferon (IFN-γ) and interleukin 12 (IL-12) secretion; however, the mechanism underlying this enhanced immunity against L. amazonensis infection remains largely unresolved. In this study, we utilized stationary promastigotes to infect bone marrow-derived dendritic cells (DCs) of C57BL/6 mice and assessed the activation of DC subsets and the capacity of these DC subsets to prime CD4⁺ T cells in vitro. We found that CXCL10 induced IL-12 p40 production but reduced IL-10 production in uninfected DCs. Yet L. amazonensis-infected DCs produced elevated levels of IL-10 despite CXCL10 treatment. Elimination of endogenous IL-10 led to increased IL-12 p40 production in DCs as well as increased proliferation and IFN-γ production by in vitro-primed CD4⁺ T cells. In addition, CXCL10-treated CD4⁺ T cells became more responsive to IL-12 via increased expression of the IL-12 receptor β₂ chain and produced elevated levels of IFN-γ. This report indicates the utility of CXCL10 in generating a Th1-favored, proinflammatory response, which is a prerequisite for controlling Leishmania infection.     

Specific CD4+ T-Cell Reactivity and Cytokine Release in Different Clinical Presentations of Leptospirosis

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4⁺ T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure to Leptospira). Whole-blood specimens were stimulated with Leptospira antigens in vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4⁺ T cells. The production of inflammatory cytokines such as TNF-α by CD4⁺ T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease.     

Porphyromonas gingivalis Fimbriae Use β2 Integrin (CD11/CD18) on Mouse Peritoneal Macrophages as a Cellular Receptor, and the CD18 β Chain Plays a Functional Role in Fimbrial Signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of β₂ integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the β chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) genes in the cells. Using a binding assay with ¹²⁵I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of β₂ integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1β and TNF-α genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1β and TNF-α genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of β₂ integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

Increased numbers and functional activity of CD56+ T cells in healthy cytomegalovirus positive subjects

Mycobacterium indicus pranii (MIP) is an atypical mycobacterial species possessing strong immunomodulatory properties. It is a potent vaccine candidate against tuberculosis, promotes Th1 immune response and protects mice from tumours. In previous studies, we demonstrated higher protective efficacy of MIP against experimental tuberculosis as compared with bacillus Calmette–Guérin (BCG). Since macrophages play an important role in the pathology of mycobacterial diseases and cancer, in the present study, we evaluated the MIP in live and killed form for macrophage activation potential, compared it with BCG and investigated the underlying mechanisms. High levels of tumour necrosis factor-α, interleukin-12p40 (IL-12p40), IL-6 and nitric oxide were produced by MIP-stimulated macrophages as compared with BCG-stimulated macrophages. Prominent up-regulation of co-stimulatory molecules CD40, CD80 and CD86 was also observed in response to MIP. Loss of response in MyD88-deficient macrophages showed that both MIP and BCG activate the macrophages in a MyD88-dependent manner. MyD88 signalling pathway culminates in nuclear factor-κB/activator protein-1 (NF-κB/AP-1) activation and higher activation of NF-κB/AP-1 was observed in response to MIP. With the help of pharmacological inhibitors and Toll-like receptor (TLR) -deficient macrophages, we observed the role of TLR2, TLR4 and intracellular TLRs in MIP-mediated macrophage activation. Stimulation of HEK293 cells expressing TLR2 in homodimeric or heterodimeric form showed that MIP has a distinctly higher level of TLR2 agonist activity compared with BCG. Further experiments suggested that TLR2 ligands are well exposed in MIP whereas they are obscured in BCG. Our findings establish the higher macrophage activation potential of MIP compared with BCG and delineate the underlying mechanism.