As a first defensive step towards any infection is the inflammatory response hoisted by the host to restrict pathogen invasion and disease spread. But in turn, the pathogen exploits and modulates the host defense mechanisms to sustain its spread and survival within the host. Activation of coagulation and fibrinolytic pathways in response to various bacterial infections are critical components elicited by both the above processes 
. Bacterial endotoxin LPS, immune complexes and many other factors elaborated in various infectious diseases are shown to induce procoagulant TF expression in monocytes/macrophages and the endothelium, which under normal healthy state do not express TF 
. Cytokine and TF expression in blood circulation in response to microbial infection is shown to contribute to septic shock, intravascular coagulation, pulmonary fibrin deposition and multi organ failure in various animal models of bacterial infection 
. Mycobacterium tuberculosis
is not a gram negative bacteria but is more related to the Nocardia and Corynebacterium
species with shorter chain fatty acids. Mycobacteria
possess a characteristic hydrophobic and waxy cell wall, rich in mycolic acids, peptidogylcan and arabinogalactan. Thus, it is quite probable that Mycobacteria
may utilize different macrophage receptors and induce distinct signaling pathways compared to other gram positive and negative organisms.
In the present study, we show that macrophages in response to Mtb infection express massive amounts of TF, in quantities much higher than the known potent stimulus, the gram-negative bacterial lipopolysaccharides. However, comparing a whole organism Mtb preparation to LPS may not be a true comparison as the higher level of TF induction in macrophages treated with γ-irradiated Mtb could be a cumulative response to multiple factors in Mtb. The increased TF activity in macrophages in response to Mtb stems from de novo synthesis of TF. The kinetics of TF mRNA and protein expression patterns and levels differ between Mtb and LPS induction, with Mtb showing somewhat delayed but robust expression that persisted for longer time periods. Although the precise molecular mechanisms and effector signaling molecules that regulate Mtb-mediated transcription and translation of TF have not been resolved, herein, we have identified that Mtb-induced TF expression in macrophages is mediated, at least in part, by TLR2, TLR4 and CD14. In contrast to LPS stimulation, where CD14 inhibition completely abrogated TF induction as well as TNF-α secretion, CD14 inhibition only minimally reduced Mtb-mediated TF induction. Blockade of TLR2 and TLR4 did not prevent TF induction by Mtb. However, inhibition of TLR2 and TLR4 along with CD14 inhibited Mtb-induced TF activity by about 50%. A partial and incomplete inhibition of Mtb-induced TF expression by blockade of all three receptors -CD14, TLR2, and TLR4 indicate that other co-receptors/co-factors present on the surface of macrophages must cooperate with CD14/TLR2/TLR4 for Mtb-mediated TF induction.
Macrophages are known to synthesize and secrete cytokines, including TNF-α, IL1-β, IL-8, IL-6, IFN-γ and others, upon infection with Mtb
. Among these, TNF-α, and IL1-β are known potent inducers of TF expression in endothelial cells 
. It had been reported that TNF-α, IL1-β and IFN-γ could also induce TF expression in macrophages, but to a lesser extent 
. Therefore, it is conceivable that TNF-α, IL1-β or IFN-γ secreted upon mycobacterial infection could be responsible for Mtb-
mediated TF induction. However, this seems unlikely as exogenous addition of recombinant TNF-α, IL1-β, IL-6 or IL-8 failed to induce TF expression in isolated macrophages in our experiments. Although IFN-γ treatment slightly increased TF activity in macrophages, the magnitude of Mtb-
induced TF expression far exceeds the effect of IFN-γ. Moreover, IFN-γ neutralizing antibodies failed to inhibit Mtb-
induced TF expression. Furthermore, conditioned media from macrophages infected with either H37Rv or H37Ra that contain macrophage secreted cytokines and probably some bacteria-derived soluble factors also failed to induce TF activity when added to fresh macrophages.
Earlier studies showed that mycobacterial components can induce TF 
. Trehalose 6, 6-dimycolate also known as cord factor is an important component modulating various macrophage functions and has been shown to induce macrophage TF and TNF-α induction 
. However, a complete analysis of other components from virulent Mtb
responsible for TF induction is unknown. In this study, we have successfully identified the various cell wall components involved in TF induction. We found that the fraction containing only cell wall components of Mtb can be
as potent as the Mtb
whole cell lysate. Further, culture filtrate proteins of Mtb
were not efficient inducers of TF expression in macrophages. These data suggest the likelihood of non-involvement of Mtb
secreted antigens in inducing TF expression. Among the purified cell wall components analyzed, mAGP complex induced the highest levels of TF activity expression but it is interesting to note that the individual components that comprise the core mAGP such as mycolic acid, arabinogalactan and peptidoglycan individually did not show TF induction indicating towards a specific conformational requirement of the mAGP complex as a pathogen-associated-molecular-pattern (PAMP) for recognition by pattern recognition receptors on macrophages such as TLRs to induce TF expression.
Considering the fact that the two mycobacterial strains of Mtb
, H37Ra and H37Rv differ at the genetic level, in macrophage activation mechanisms, cytokine elaboration and display completely distinct disease pathology 
, we expected that they may show differential potential for TF induction. However we found only minimal differences between these two strains in their ability to induce TF expression. It is possible that both the strains may have relatively similar composition but may have varied abundance or vice-versa
of some of the outer cell wall components that are responsible for TF induction.
Coagulation and inflammation share a bidirectional relation playing crucial roles in host defense 
. Proinflammatory cytokines produced in infections can induce TF expression on monocytes and endothelial cells 
. In turn, TF, in addition to activating coagulation, can also amplify the production and release of inflammatory mediators by signaling mediated by TF-FVIIa complex formation and downstream proteases generated by TF-FVIIa-induced coagulation 
. Ample studies conducted have provided strong evidences that by blocking TF, the proinflammtory cytokine elaboration (IL-6, IL-8), levels of soluble TNF receptor-1 and infection-associated organ injury and mortality were reduced 
. It will be interesting to examine in future whether Mtb
-induced TF plays a role in propagation of inflammatory responses induced by Mtb
Most of the studies that investigated the potential role of TF in modulating inflammation and vice-versa
were limited to acute infection and inflammatory settings 
. Our present study indicates a potential role of TF in chronic infection/inflammation setting. Tuberculosis is characterized by the presence of a granulomatous lesion. It is known that later stages of granuloma formation are accompanied by fibrosis which forms a barrier and have important implications in containing the spread of infection and inflammation 
. It is thus possible that localized activation of coagulation by TF expression on the surface of activated lung macrophages in response to Mtb
infection would lead to thrombin generation and subsequently fibrin deposition required for stable granuloma formation. In a recent study, using fibrinogen-knockout mice treated with mycobacterial trehalose dimycolate, it was shown that fibrinogen was required for proper granuloma formation 
. In other studies, fibrinogen-deficient mice and wild-type mice treated with warfarin to suppress fibrin formation were found to display increased mortality upon peritoneal infection with Listeria monocytogenes
, indicating the importance of fibrin in restricting the infection 
. In agreement with these above findings, a recent study showed impaired cytokine and chemokine production, suppressed neutrophil recruitment, increased hepatic bacterial burden and mortality in fibrinogen-deficient mice following infection with Yersinia enterocolitica
. In the same study, mice with low TF activity succumbed to yersiniosis with a phenotype similar to fibrin(ogen)-deficient mice, indicating that the extrinsic coagulation pathway led to the protection.
In the absence of structurally intact granuloma formation, the bacteria may spread from the localized foci following macrophage apoptosis that results from increased bacterial burden. This phenomenon could further lead to systemic infection and inflammation through activation of endothelium either by the bacteria itself or by TF-bearing microparticles produced by activated and apoptotic macrophages 
. Endothelial cells could actively contribute to host's innate immune response against mycobacteria. Earlier studies reported that endothelial cells are susceptible to mycobacterial infection and influence bacterial replication, dissemination and elimination 
. These evidences along with our data showing prolonged endothelial induction of TF expression upon Mtb
infection provides clues as to how an initial localized pulmonary infection could transform into a systemic infection. Increased TF expressing macrophages, TF-bearing microparticles and procoagulantly active endothelium could thus serve as a platform for increased thrombus formation with consequences such as DIC and DVT. However, further studies are needed in future to demonstrate the relevance of TF expression in tuberculosis disease pathology employing appropriate animal models.