Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation

Acknowledgements We thank Lizette Fick for her contribution to histopathology. We thank Faried Abbass for technical support. We thank the support staff of the Division of Immunology and the Research Animal Facility at the University of Cape Town for their contribution to animal care and technical support. The study was supported by the University of Cape Town, National Research Foundation (South Africa), South African Medical Research Council (SAMRC) National Health Laboratory Service (South Africa), The European Union (contract number: 028190), FP6 NEST project N°028190 “TB REACT”. Research carried out within the scope of the Franco/South African Laboratory “TB Immunity” (Associated International Laboratory ‘AIL’).Peer reviewedPublisher PD

CD14 Works with Toll-Like Receptor 2 to Contribute to Recognition and Control of Listeria monocytogenes Infection

Toll-like receptor 2 (TLR2) signaling has been shown to contribute to resistance to Listeria monocytogenes infection, as TLR2-deficient mice have a heightened susceptibility to infection with this organism. Because CD14 may associate with TLR2, we investigated the role of CD14 in Listeria responses. In both CD14-deficient and TLR2- deficient macrophages, nuclear factor κB translocation; CD40 and CD86; and the production of interleukin (IL)- 12, IL-6, tumor necrosis factor, and nitric oxide are reduced. The absence of CD14 augmented susceptibility to Listeria infection, reduced survival, and diminished bacterial clearance, as observed in TLR2-deficient mice.Compared with C57BL/6 control mice, CD14-deficient mice were observed to have a greater number of hepatic microabscesses containing abundant neutrophils, these abscesses were larger in size, and there was reduced inducible nitric oxide synthase expression. Further, mice that are both CD14 deficient and TLR2 deficient display susceptibility to infection that is comparable to that of mice deficient in either CD14 or TLR2 alone. Therefore, the present data demonstrate the role of CD14 and TLR2 in the recognition and control of Listeria infection and host resistanc

Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM1 isomer and PIM2 mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM1 and PIM2 analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM1 and PIM2 analogues. CD14 was dispensable for PIM1 and PIM2 analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM1 and PIM2 analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway

Reactivation of M. tuberculosis Infection in Trans-Membrane Tumour Necrosis Factor Mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established

All-Trans Retinoic Acid Promotes TGF-β-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus

It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4(+)Foxp3(+) regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.Addition of atRA to naïve CD4(+)CD25(-) cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3(+) iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4(+) cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4(+) cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4(+) cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4(+) cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation

Cell iron status influences macrophage polarization.

Macrophages play crucial roles in innate immune response and in the priming of adaptive immunity, and are characterized by their phenotypic heterogeneity and plasticity. Reprogramming intracellular metabolism in response to microenvironmental signals is required for M1/M2 macrophage polarization and function. Here we assessed the influence of iron on the polarization of the immune response in vivo and in vitro. Iron-enriched diet increased M2 marker Arg1 and Ym1 expression in liver and peritoneal macrophages, while iron deficiency decreased Arg1 expression. Under LPS-induced inflammatory conditions, low iron diet exacerbated the proinflammatory response, while the IL-12/IL-10 balance decreased with iron-rich diet, thus polarizing toward type 2 response. Indeed, in vitro macrophage iron loading reduced the basal percentage of cells expressing M1 co-stimulatory CD86 and MHC-II molecules. Further, iron loading of macrophages prevented the pro-inflammatory response induced by LPS through reduction of NF-κB p65 nuclear translocation with decreased iNOS, IL-1β, IL-6, IL-12 and TNFα expression. The increase of intracellular iron also reduced LPS-induced hepcidin gene expression and abolished ferroportin down-regulation in macrophages, in line with macrophage polarization. Thus, iron modulates the inflammatory response outcome, as elevated iron levels increased M2 phenotype and negatively regulated M1 proinflammatory LPS-induced response

Iron loading of primary macrophages impairs LPS-induced pro-inflammatory responses.

<p>Bone marrow derived-macrophages were preincubated with ferric ammonium citrate (FAC, 0, 50 or 100 μM) overnight followed by LPS stimulation (100 ng/mL) for 4 h or 24 h to assess <i>Il-12b</i>, <i>Il-1β</i>, <i>Il-6</i>, <i>Tnfα</i> and <i>iNos</i> mRNA gene expression by quantitative PCR analysis (<b>A</b>) or cytokine Il-12p40, Il-1β, Il-6, Tnfα and nitric oxide measurement in cell medium by ELISA and Griess assay (<b>B</b>), respectively. Bone marrow derived-macrophages were preincubated with ferric ammonium citrate (FAC 100 μM) or medium overnight followed by LPS stimulation (100 ng/mL) for 30 min to assess NF-κB nuclear translocation by immunofluorescence, with NF-κB p65 in green and DAPI in red (<b>C</b>). Data of mRNA gene expression are given as fold change gene expression relative to the expression in untreated cells. Data are representative of at least two independent experiments and presented as mean ± SD (n = 4). * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.</p

Expression of macrophage polarization markers upon iron overload in liver, spleen and peritoneal macrophages.

<p>C57BL/6 wild type mice were fed with iron replete diet (IR), or iron-rich diet for 3 days (Fe diet) or received an injection of iron-dextran (Fe-dextran, 0.2 g/kg ip) for 48h. Liver homogenates (<b>A</b>), spleen (<b>B</b>) and peritoneal exudate macrophages (<b>C</b>) gene expression of macrophage polarization marker <i>Arginase-1 (Arg1)</i>, <i>Chitinase-like 3 (Ym1)</i>, CD206 mannose receptor (<i>Mrc</i>) or <i>iNos</i> was analyzed using quantitative PCR analysis. Data are expressed as mRNA fold change relative to control mice fed with IR diet. Data are from two independent experiments and presented as mean ± SD (n = 6 mice per group). ns, non significant; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.</p

Long-Term Control of Mycobacterium bovis BCG Infection in the Absence of Toll-Like Receptors (TLRs): Investigation of TLR2-, TLR6-, or TLR2-TLR4-Deficient Mice

Live mycobacteria have been reported to signal through both Toll-like receptor 2 (TLR2) and TLR4 in vitro. Here, we investigated the role of TLR2 in the long-term control of the infection by the attenuated Mycobacterium, Mycobacterium bovis BCG, in vivo. We sought to determine whether the reported initial defect of bacterial control (K. A. Heldwein et al., J. Leukoc. Biol. 74:277-286, 2003) resolved in the chronic phase of BCG infection. Here we show that TLR2-deficient mice survived a 6-month infection period with M. bovis BCG and were able to control bacterial growth. Granuloma formation, T-cell and macrophage recruitment, and activation were normal. Furthermore, the TLR2 coreceptor, TLR6, is also not required since TLR6-deficient mice were able to control chronic BCG infection. Finally, TLR2-TLR4-deficient mice infected with BCG survived the 8-month observation period. Interestingly, the adaptive response of TLR2- and/or TLR4-deficient mice seemed essentially normal on day 14 or 56 after infection, since T cells responded normally to soluble BCG antigens. In conclusion, our data demonstrate that TLR2, TLR4, or TLR6 are redundant for the control of M. bovis BCG mycobacterial infection

Iron status in liver, spleen and peritoneal macrophages.

<p>C57BL/6 wild type mice were fed with iron replete diet (IR), or iron-rich diet for 3 days (Fe diet) or received an injection of iron-dextran (Fe-dextran, 0.2 g/kg ip) for 48h. (<b>A</b>) Body weight, relative liver and spleen weights are indicated. (<b>B</b>) Representative DAB-enhanced Perl’s staining of liver, spleen, and peritoneal exudate cells spin show iron deposit (in brown). (<b>C</b>) <i>Hepcidin 1 (Hamp1</i>) and <i>ferritin L</i> (<i>Ftl</i>) gene expression in liver tissue homogenates, spleen and peritoneal cells was analyzed using quantitative PCR. Data are expressed as mRNA fold change relative to control mice fed with IR diet. Data are from two independent experiments and presented as mean ± SD (n = 6 mice per group). ns, non significant; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.</p