Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection: Innate Immunity

Tissue Factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TFΔ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2 like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth

NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection

In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice

Mycobacterium tuberculosis infection and tissue factor expression in macrophages.

A number of earlier studies reported the occurrence of thrombotic complications, particularly disseminated intravascular coagulation and deep vein thrombosis, in tuberculosis (TB) patients. The aberrant expression of tissue factor (TF), the primary activator of coagulation cascade, is known to be responsible for thrombotic disorders in many diseases including bacterial infections. Further, expression of TF by cells of the monocyte/macrophage lineage is also shown to contribute to the development and progression of local and systemic inflammatory reactions. In the present study, we have investigated whether Mycobacterium tuberculosis (Mtb) infection induces TF expression in macrophages, and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. The data presented in the manuscript show that both live virulent Mtb and γ-Mtb treatments markedly increased TF activity in macrophages, predominantly in the CD14(+) macrophages. Detailed studies using γ-Mtb showed that the increased TF activity in macrophages following Mtb treatment is the result of TF transcriptional activation. The signaling pathways of TF induction by Mtb appears to be distinct from that of LPS-induced TF expression. Mtb-mediated TF expression is dependent on cooperation of CD14/TLR2/TLR4 and probably yet another unknown receptor/cofactor. Mtb cell wall core components, mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) were identified as factors responsible for induction of TF in the order of mAGP>PIM6>LM. A direct contact between bacteria and macrophage and not Mtb-released soluble factors is critical for TF induction by Mtb. In summary, our data show that Mtb induces TF expression in macrophages and Mtb signaling pathways that elicit TF induction require cooperation of multiple receptors, co-receptors/co-factors including Toll-like receptors. The importance of TF in granuloma formation and containment of Mtb is discussed

Memory like NK cells display stem cell like properties after Zika virus infection.

NK cells have been shown to display adaptive traits such as memory formation akin to T and B lymphocytes. Here we show that Zika virus infection induces memory like NK cells that express CD27. Strikingly, these cells exhibit stem-like features that include expansion capacity, self-renewal pathway, differentiation into effector cells, longer telomeres and gene signature associated with hematopoietic stem cell (HSC) progenitors. This subset shared transcriptional and epigenetic changes with memory CD8 T cells, stem cells and stem like T cells. These NK cells with memory and stem cell features, which we term "NK memory stem cells", demonstrated greater antiviral potential than CD27- or naïve CD27+ NK when adoptively transferred to Zika infected mice. Our results also suggest a role for the transcription factor TCF-1 in memory and stemness features of this NK subset. This study defines a unique TCF1hi CD27+ NK subset with memory capacity and stem cell features that play a role in antiviral immunity

TLR2 and TLR4 along with CD14 receptor are partly responsible for <i>γ-Mtb</i>-mediated TF induction.

<p>(<b>A</b>) MDMs were treated with TLR2 agonist (Pam3CSK4, 1 µg/ml), TLR4 agonist (MPLA, 1 µg/ml) and TLR9 agonist (ODN2006, 10 µg/ml) for 9 h. As a control, MDMs were also treated with γ-<i>Mtb</i> H37Rv (10 µg/ml). Cell surface TF activity was determined in factor X activation assay.* indicates significant difference compared to untreated cells (P≤0.01); # denotes significant difference compared to γ-<i>Mtb</i> treated cells (P<0.01) (<b>B</b>) MDMs were preincubated with CD14, TLR2 and TLR4 blocking antibodies (each, 10 µg/ml) either alone or in combination and with isotype control IgG for 45 min at 37°C and then cells were stimulated with γ-<i>Mtb</i> for 9 h. At the end of the stimulation period cell surface TF activity was measured in factor X activation assay. # denotes significant inhibition compared to γ-<i>Mtb</i> or LPS alone treated cells (P<0.05); paired t-test. Data are mean ± SEM (n = 3–8).</p

ERK and PKC pathways play a role in <i>Mtb</i>-induction of TF procoagulant activity.

<p>MDMs were pretreated with the MEK inhibitors, PD98059 and U0126 and PKC inhibitors, GF109203 and Gö6983, p38 MAPK inhibitor, SB203580 and PI3-AKT inhibitors, LY294002 and wortmannin (all used at 10 µM concentration) for 1 h. Cells were then stimulated with γ-<i>Mtb</i> (10 µg/ml) for 9 h. Cell surface TF activity measured in factor X activation assay. Values obtained for γ-<i>Mtb</i> alone treated cells are represented as 100%. # indicates significant inhibition compared to γ-<i>Mtb</i> alone treated cells (P = 0.0001). Data are mean ± SEM ( n = 7).</p

Both H37Ra and H37Rv induce TF on macrophages.

<p>MDMs in complete medium without antibiotic were infected with live H37Rv and H37Ra at varying ratios of bacteria∶cell. After 2 h, the cells were washed twice with warm RPMI serum free medium and then incubated overnight in fresh complete medium. At the end of incubation, TF activity at the cell surface was measured in factor X activation assay. ns, not statistically significant. Data are mean ± SEM ( n = 5).</p

<i>γ-Mtb</i> induces TF expression in endothelial cells which follows different kinetics than TNF-α+IL1-β.

<p>HUVEC were stimulated with TNF-α+IL1-β (20 ng/ml each) (open circles) or <i>γ</i>-<i>Mtb</i> H37Rv (10 µg/ml) (closed circles) in EBM-2 complete medium for varying time periods. At the end of the treatment, TF activity on intact monolayers was measured in factor X activation assay. * denotes significant difference from untreated control for all time points (P<0.001). Data are mean ± SEM (n = 3).</p