This leads us to speculate that with tools of the appropriate sen

This leads us to speculate that with tools of the appropriate sensitivity,

one should be able to find a large number of autoreactive T cells, even in a normal repertoire, maintained in a tolerant state by nondeletional mechanisms. Mice from the NIAID contract facility (Taconic Farms, Germantown, NY, USA) were housed pathogen free. B10.A CD45.2 mice were also crossed to B6,CD45.1 mice to generate a B10.A,CD45.1 strain [20]. To generate B10.A, mPCC(tg),CD45.1 mice, B10.A mPCC-transgenic, CD45.2 mice [19] were bred to B10.A,CD45.1. The IEk restricted MCC (Moth Cytochrome C)/PCC specific TCR transgenic 5C.C7 mice on Rag2−/−, CD45.1+/+, and CD45.2+/+ backgrounds have been previously described [5]. A1(M) mice originally from Steve Cobbold find more [21] on a CBA/Ca background were backcrossed 11 times onto a B10.A,Rag2−/− background [14] and maintained by homozygous breeding. All animal protocols were as approved by the NIAID animal care and use committee. For adoptive cell transfers, cell suspensions from pooled lymph nodes of donor TCR-Tg Rag2−/− mice (>90% CD4+ T cells) were used without further enrichment and injected by the suborbital route. Acute antigen challenges were performed by intraperitoneal

injections of 30 μg of antigenic peptide (DbY or PCC; Anaspec or Bachem, USA) mixed with 5 μg of LPS (Sigma, MI, USA). T cells in transfer recipients were enumerated by isolating all lymph nodes and spleen, chopping them to approximately 1 mm cubes and digesting check details with 2 mg/mL collagenase-D (Roche, USA) solution containing 3 mM CaCl2 in 1× PBS, at 37°C for 45 min. Digested tissue was dissociated using gentleMACS dissociator and gentleMACS dissociator C tubes (Miltenyi biotec, Germany) with manufacturer’s programmed settings m_Spleen 2.01 followed by m_Spleen 3.02 run serially on each sample. A total of Sulfite dehydrogenase 500 μL aliquots of the single cell suspensions were stained to obtain the percentage of CD4+ T cells and used to calculate the number of CD4+ T cells in each animal without any further manipulation. However, in order to track exceedingly low numbers

of transferred T cells, further enrichment was necessary. Following absolute counts, as stated above, as remaining cells were washed and centrifuged over Ficoll-Paque PLUS (GE Healthcare Bioscience) followed by enrichment for T cells by negative selection. Briefly, a cocktail of mouse and rat antibodies to B220 (RA3-6B2), CD11b (M1/770), I-EK (14.4.4s), CD8 (53-6.7), and MHC II (M5.114) (BD Bioscience) were used to label the cells and the bound fraction, pulled out using anti-mouse IgG and anti-rat IgG coated Dynabeads (Dynal Invitrogen). T cells were analyzed on a FACS Canto II cytometer (BD Immunocytometry) after staining with appropriate fluorophore coupled antibodies (Biolegend, Ebioscience or BD). We thank Eleanore Chuang for assistance with experiments, and Pascal Chappert for discussions. This research was supported by the Intramural Research Program of the NIH, NIAID.

CD103+ DCs display an enhanced capacity to produce RA [26], high

CD103+ DCs display an enhanced capacity to produce RA [26], high expression of IDO [27], thymic stromal lymphopoietin- [28] and β8-integrin-mediated activation of TGF-β [29]. Thus LP-derived DCs in the mLNs through various mechanisms support the efficient conversion of conventional T cells into iTreg cells. Besides their ability to foster iTreg-cell generation, intestinal CD103+ DCs are imprinted with an enhanced capacity to induce the gut-homing molecules β7-integrin and CCR9 in activated T cells

[25, 26]. Yet, in vivo induction of gut-homing potential in such cells required additional factors that were provided by nonhematopoietic stroma cells [30]. We observed that BM-derived DCs fail to support gut-homing molecule induction in vitro, but can do so in vivo when injected into mLN afferent lymphatics. Conversely, SCH727965 in vitro endogenous LP-derived check details DCs failed to induce gut-homing molecules in lymph node grafts of peripheral origin [30]. This indicates that in vivo non-DC-dependent factors contribute to the quality of the T-cell response (reviewed in [31]). We may conclude that the microenvironment of mLNs and the unique properties of intestinal DCs synergize to enable the efficient generation of iTreg cells and a gut-homing signature on these cells. Despite the previous findings regarding the generation of iTreg cells in the mLNs, such iTreg-cell

generation still seems insufficient to generate intestinal tolerance. Instead, Selleckchem Vorinostat we found that tolerance to the model antigen OVA requires gut homing of iTreg cells and a subsequent local modulation of the Treg cells in the LP [23] (Fig. 1; for a recent review on oral tolerance refer to

[32]). As described in “iTreg-cell generation in the mesenteric lymph nodes”, gut homing requires the β7-integrin, which binds to its ligand MadCAM-1 that is expressed by gut venules. Consistently, β7-integrin-deficient mice fail to generate tolerance to OVA and this defect can be rescued by the adoptive transfer of β7-competent OVA-specific T cells in WT but not MadCAM-1-deficient mice [23]. Within the gut LP, iTreg cells proliferate vigorously and macrophage-dependent signals enable a shift in the overall ratio of Foxp3+ to Foxp3− cells in favor of Treg cells. Thus the gut LP takes an active role in shaping the Treg-cell pool by expanding iTreg-cell populations, which also explains why the TCR repertoire of gut Treg cells differs from that of Treg cells of other origins. Notably, we observe Treg cells in the afferent lymph connecting the intestine to the mLNs, thus documenting that these cells can travel back to their place of birth (O. Pabst, unpublished observation). Interestingly, there is evidence that Treg-cell populations might be modulated in other tissues as well. In skin-draining LNs the frequency of skin-derived Treg cells increases after inflammation [33] and, in an allograft model, Treg-cell-mediated suppression requires the migration of Treg cells from the graft to the draining LNs [34].

Subsequent investigations have suggested that vitamin D, via cath

Subsequent investigations have suggested that vitamin D, via cathelicidin, can also induce autophagy One study has shown that vitamin D3 specifically induces autophagy in human monocytes and macrophages via cathelicidin [49], and that cathelicidin comes into direct contact with mycobacteria within the autophagosome. Vitamin D supplementation in patients deficient in vitamin D did not, however, increase circulating cathelicidin [50]. None the less, localized increases of this anti-microbial peptide may be achievable in the granuloma – which might not be detectable by peripheral sampling. Further studies are needed to

assess the true benefits, if any, of vitamin D in the immune response to tuberculosis and what role Compound Library cost autophagy might play in this. Autophagy assists with antigen processing of intracellular and extracellular material for major histocompatibility complex (MHC) class I and class II presentation, and has also been shown to Roxadustat order be important for efficient cross-presentation to CD8+ T lymphocytes. Autophagosomes containing pathogens, including mycobacteria, converge with endosomes and thus deliver antigens for loading in MHC class II compartments. Autophagy can also deliver endogenous antigens to the MHC II pathway [51] enhancing presentation to CD4+ T cells [52–56]. These studies showed a direct association of autophagy

with enhanced delivery of endogenous proteins to the MHC class II pathway and suggest that autophagy is a mechanism by which the peptide repertoire presented by MHC class II molecules may be extended from exogenous to endogenous antigens.

Methisazone There is evidence that autophagy-associated proteins, including LC3, gain access to MHC II compartments [57] and coupling of antigens to Atg8/LC3 enhanced their presentation on MHC class II [58]. Moreover, the induction (with rapamycin or starvation) or suppression (with 3-MA or RNAi knock-down) of autophagy have been shown to have direct effects on MHC II-peptide presentation [59,60]. In vivo, autophagy has also been shown to be important for MHC class II presentation of self-proteins during central tolerance induction [61]. In the context of mycobacteria, autophagy also enhances MHC class II presentation. Vaccination with rapamycin-treated DC enhanced MHC class II presentation of Ag85B and was associated with the induction of potent protective CD4+ responses in mice [62]. Autophagy may also contribute to the generation of MHC class I-restricted responses. English et al. demonstrated that autophagy contributed to processing of herpes simplex virus-1 antigens for MHC class I presentation [63]. Autophagy may also influence antigen presentation to CD8+ T cells via degradation of the MHC class I molecules themselves [64]. Autophagy induction resulted in reduced MHC class I surface expression, consistent with the presence of MHC I in autophagosomes, but this was reversed by IFN-γ.

19–22 Infection with Listeria monocytogenes in mice is a widely u

19–22 Infection with Listeria monocytogenes in mice is a widely used experimental model for identifying the immune mediators of innate and adaptive host defence against intracellular bacterial pathogens.23–25 Interferon-γ produced by NK and both CD4+ and CD8+ T-cell subsets each play important roles in innate host defence at early time-points after this infection.26–29 At later infection time-points, the

INK 128 molecular weight expansion of L. monocytogenes-specific CD8+ and CD4+ T cells coincides with bacterial eradication, and thereafter the absolute numbers of pathogen-specific cells contract, and are maintained at ∼ 5 to 10% of peak expansion levels.24,25 During secondary infection, L. monocytogenes-specific T cells re-expand and rapidly confer sterilizing immunity to infection. Although the cellular mediators that confer protection in each phase of L. monocytogenes infection have been

identified, the specific cytokine signals that activate and sustain these cells remain largely undefined. Given the potency whereby IL-21 stimulates the activation of NK, Roxadustat CD8+ and CD4+ T cells, and the importance of these cells in host defence against L. monocytogenes, the requirement for IL-21 in innate and adaptive immunity after this acute bacterial infection was examined in this study. Interleukin-21-deficient mice on a C57BL/6 (B6) background were obtained from Dr Matthew Mescher through Lexicon Genetics and the Mutant Mouse Regional Resource Centers. B6 control mice were purchased from the National Cancer Institute (Bethesda, MD). Mice with individual defects in IL-12P40 or type I IFN receptor, and mice with combined defects in both IL-12P40 and type I IFN receptor (i.e. double

knockout; DKO) have been described.30,31 Mice with combined defects in IL-21, IL-12, and type I IFN receptor (triple knockout; TKO) were generated by inter-crossing IL-21-deficient mice with type I IFN receptor-deficient mice, and then inter-crossing these mice with DKO mice. All experiments were performed under University of Minnesota Institutional Animal Care and Use Committee approved protocols. The wild-type L. monocytogenes strain 10403s, recombinant L. monocytogenes ovalbumin (Lm-OVA), and recombinant Lm-OVA ΔactA that allow a more precise analysis of the immune response to the surrogate L. monocytogenes-specific H-2Kb OVA257–264 antigen have each been described.30–32 For infections, L. monocytogenes was grown to early log phase (optical density at 600 nm 0·1) in brain–heart infusion medium at 37°, washed, and diluted with saline to 200 μl final volume and injected intravenously. At the indicated time-points after infection, the number of recoverable L. monocytogenes colony-forming units (CFUs) in the organs of infected mice were quantified by homogenization in saline containing Triton-X (0·05%), and plating serial dilutions of the homogenate on agar plates as described.

2) These data suggest that STUB1 is required for T-cell activati

2). These data suggest that STUB1 is required for T-cell activation, and it plays a role in the upstream of TAK1 and the IKK signalsome in TCR-NF-κB signaling pathway. In order to determine the target of STUB1, we examined the association between STUB1 and main components involved in TCR signaling by Co-IP. The results showed that overexpressed STUB1 interacted with MALT1, TRAF6, TAK1, and IKK-α, but not with BCL10, IKK-β, or IKK-γ (Fig. 2A and Supporting Information Fig. 3). Interestingly, altering the expression level of STUB1 by RNAi-mediated knockdown did not affect

the total expression levels of Temozolomide solubility dmso all these signal proteins markedly, indicating that STUB1 catalyzes the target protein by a nonlytic ubiquitin modification (Supporting Information Fig. 4). The STUB1-associated molecules were then cotransfected with ubiquitin to check whether their ubiquitination status was affected by STUB1 or not. As shown in Fig. 2B, cotransfection

with STUB1 caused a mass of ubiquitination of CARMA1, and moderate ubiquitination of MALT1. In contrast, ubiquitination of other STUB1-associated proteins, such as TRAF6, TAK1, and IKK-α, were not markedly altered by STUB1 (Fig. 2C), suggesting that STUB1 specifically catalyzes the ubiquitination of CARMA1 and MALT1. We next challenged stable Jurkat E6 cells expressing STUB1-RNAi or controls Protein Tyrosine Kinase inhibitor with P/I, and performed Co-IP and immunoblot Chorioepithelioma to determine the effects of STUB1 on endogenous ubiquitination of CARMA1 and MALT1 upon stimulation. The results showed that the ubiquitination of CARMA1 in control cells was induced at the early phase by P/I stimulation, and it was significantly impaired in STUB1-knockdown cell (Fig. 2D), suggesting that STUB1 is essential for P/I-induced CARMA1 ubiquitination. In contrast, the ubiquitination of MALT1 by P/I stimulation was not markedly affected by STUB1-knockdown, and the basic level of MALT1 ubiquitination in STUB1-RNAi-transfected cells was higher than that in control cells (Fig. 2E). The above results suggest that STUB1 facilitates

TCR signaling by specifically catalyzing the ubiquitination of CARMA1. To examine how STUB1 affected the ubiquitination of CARMA1 in detail, we first determined the minimal regions of CARMA1 responsible for its interaction with STUB1 by Co-IP. A series of truncation mutation expression plasmids of CARMA1 was constructed and used. The results showed that the PDZ (aa 661-742) and SH3 (aa 766-834) domains of CARMA1 were responsible for the interaction with STUB1 (Fig. 3A). MAGUK region of CARMA1, containing PDZ, SH3, and GUK domains, not only functions in localizing and clustering multiprotein signaling complexes on the cell membrane, but also controls the ubiquitination of CARMA1 [6, 18]. We then mutated each of all seven lysine residues at the PDZ and SH3 domains, and performed ubiquitination assays.

Human waste, bed pans and urinals should be placed, handled, stor

Human waste, bed pans and urinals should be placed, handled, stored/disposed of separately in time and space to other items, particularly food.[9] Attempting to correctly pronounce Māori names is polite and appropriate. In the words of another Māori proverb: Ki mai ki ahau, he aha te mea nui o te ao, māku e kii atu – He Tangata, He Tangata, He Tangata. When I am asked what is the greatest treasure on earth I will reply – it is the people, it is the people, it is the people. Steven May Patients in rural areas are both economically and medically disadvantaged. Access to specialist services in rural areas is limited. More care is likely to be out-sourced

to local physicians, GPs and palliative Maraviroc cost care nurses who

will need ‘on the ground’ outreach support from renal/palliative care services. Referral to these services may low due to knowledge of availability and previous exposure of the referring physician to the use of these services. Developments in information technology are likely to play a significant role in management (telemedicine), education and advice in these specialist areas. For the purpose of this position statement rural is defined as areas outside of the major cities. In Australia approximately one third of the population live in rural areas ( Fig. 1). The Accessibility/Remoteness Index for Australia (ARIA) is used to define rural and remote but it has significant inequities and is not supported by the Rural Doctor Association for resource allocation. Although the medicine is similar in rural and urban environments the Forskolin ic50 application is different in rural settings. The

challenges involved in organizing specialist care palliative care to rural areas compared with major urban areas relate to differences in environment especially population density and distances, infrastructure and resources. Palliative care services have generally developed in major population centres. Rural areas are characterized by a lack of specialist and well organized palliative care services. Palliative care in rural areas is generally delivered by primary care physicians and community nurses and not palliative care specialists. Renal palliative care potentially involves a further skill set that may not be in the general practitioners or even all palliative care specialists’ tool boxes. In a review of studies in rural palliative care Evans et al.[1] found that access to specialized palliative care services is a problem,[2-4] that rural patients reportedly were less likely than their urban counterparts to receive care from a hospice service,[5] that families and professionals have difficulties in accessing information[6, 7] and that communication difficulties can occur between primary care and specialists.

In addition, we note that sensitization alone, without adoptive t

In addition, we note that sensitization alone, without adoptive transfer of iNKT cells, induces a partial but significant reconstitution of CS in PD0325901 ic50 comparison with baseline, suggesting that iNKT cell–independent pathways may also exist (Groups B and E, Fig. 4A). We next asked whether CS is dependent upon any other trait of the hepatic environment other than CD1d-expressing cells. We explored the possibility of peripheral activation of iNKT cells following adoptive transfer. We investigated whether transferred hepatic iNKT cells exhibit tropism to

the livers of the recipient mice and again tested whether this might be dependent upon hepatocyte CD1d expression. We transferred activated iNKT cells into sensitized Jα18−/− and CD1d−/− mice (as mentioned earlier) and monitored by flow cytometry the percentage of hepatic T cells that were iNKT cells 1 day later. (This is the time point at which mice are challenged on the ears after adoptive transfer in our protocol.) We compared this to the percentage of iNKT cells in wild-type BALB/c mice, in which NKT cells comprised approximately 70% of hepatic T cells. In contrast, there is no evidence of re-population of donor iNKT cells into recipient livers: iNKT cells constituted <1% of total hepatic T cells in both iNKT cell–deficient strains following adoptive transfer (Fig. 4B). Navitoclax Had donor iNKT cells migrated

to recipient livers, and if this had been dependent upon hepatocyte

CD1d expression, then a difference would have been seen between the Jα18−/− and CD1d−/− mice. Furthermore, there does not appear to be any essential component of the hepatic environment other than CD1d-expressing cells, as the result was equivalent in Jα18−/− and CD1d−/− mice following adoptive cell transfer. Although this experiment demonstrates that peripheral hepatocyte-independent activation of iNKT cells may FAD occur, it remains unclear whether the suggestion of extrahepatic iNKT cell activation via CD1d–lipid complexes is merely an artefact of the artificial experimental design or whether this finding is relevant to wild-type mice. It is clear that reconstituted iNKT cell–deficient mice, despite their equivalent CS reactions, differ in the distribution of iNKT cells. The livers of reconstituted mice are not equivalent to those of wild-type mice. Certainly, in wild-type mice, iNKT cells may interact with hepatocytes via CD1d; we simply show here that such an interaction is not critical in mounting a full CS reaction. We demonstrate here that soon after contact sensitization, stimulatory lipids accumulate in the liver and facilitate the activation of iNKT cells in a CD1d-dependent manner. Remarkably, a significant increase in stimulatory capacity was seen within 30 min of sensitization.

We observed that neutrophils isolated from seven of 10 healthy do

We observed that neutrophils isolated from seven of 10 healthy donors produced a significant

amount of IL-8 in the presence of CpG-ODN without pretreatment. On the other hand, Hayashi et al. worked with isolated neutrophils from three healthy individuals; therefore, the significance of the obtained results may require additional evaluation. Furthermore, our results are consistent with other previous studies showing that human neutrophils respond to bacterial DNA (CpG DNA) with secretion of IL-8 without any pretreatment (34,35). Studies by Alvarez et al. (35) showed that bacterial DNA induces neutrophil activation such as IL-8 secretion through Selleck Palbociclib a TLR9-independent and MyD88-dependent pathway. Accordingly, our experiment showed that pretreatment of neutrophils with GM-CSF, as inducer of TLR9 expression, did not induce IL-8 after stimulation with CpG-ODN class A; therefore, it may be suggested that the IL-8 induction in neutrophils by CpG-ODN seen here is TLR9 independent. Certainly, to formally show this issue, blocking of TLR9 in human neutrophils would be required. CpG-ODN class A and B, on their own, even at high concentrations (40 μg/mL), did not lead to the release of TNF-α. The data confirm the result of previous studies demonstrating that both CpG-ODN and CpG DNA do not trigger a CpG-dependent release of this

cytokine in human neutrophils (34). The reason why a considerable amount of TNF-α is not detectable after selleck inhibitor stimulation with CpG-ODNs may be related to the low level of this cytokine in neutrophil supernatant making its detection difficult. Mature neutrophils in circulation show few ribosomes and endoplasmic reticulum structures and have, therefore, only limited capacity for protein synthesis. Consequently,

neutrophils make fewer molecules of a given cytokine than do macrophages or lymphocytes (36,37). Furthermore, it may be speculated that the activation of human neutrophils by CpG-ODN is dependent on leucocyte interactions, which cannot be reproduced in an isolated cell culture, or would require additional stimuli. Previous reports Doxacurium chloride indicated an increase in neutrophil functions after GM-CSF treatment. In addition, recently, a synergy between GM-CSF and TLRs, including TLR2 and 9, has been shown (23,38). Beside increased receptor expression, other effects such as activation of signalling molecules also play a role in TLR/GM-CSF synergy (23). In this context, GM-CSF as an inducer of TLR9 expression in neutrophils may serve to improve recognition of CpG-ODN and consequently act as a co-stimulator (23). The obtained results, here, show that co-stimulation of neutrophils with CpG-ODN class A and GM-CSF induces significant level of TNF-α production. Lately, it has been shown that GM-CSF enhances neutrophil responses induced by bacterial DNA in a CpG-independent pathway by increasing the activation of the MAPKs p38 (39).

These results open for further studies to elucidate the immunoreg

These results open for further studies to elucidate the immunoregulatory role of BMPs in B cells. CD40L and Enhancer for Ligand were from Alexis Biochemicals, Enzo Life Sciences (NY, USA). Recombinant human (rh) IL-21 was from Invitrogen (CA, USA), whereas the following recombinant proteins and Abs were purchased from R&D Systems (MN, USA): rhBMP-2, Pembrolizumab in vivo rhBMP-4, rhBMP-6, rhBMP-7, mouse rNoggin and anti-human BMP-6 mAb (clone 74219). The following biotinylated Abs were from R&D Systems: anti-activin RIA, anti-BMPR-IA, anti-BMPR-IB, anti-BMP-RII, anti-activin RIIA, anti-activin

RIIB. Streptavidin PE, anti-CD5 PECy7, anti-CD19 FITC, anti-CD19 PE, anti-CD20 allophycocyanin-H7, anti-CD20 PerCPCy5.5, anti-CD27 allophycocyanin, anti-CD27 PE, anti-CD38 FITC and anti-IgD PerCPCy5.5 were from BD (CA, USA), anti-CD19 Quizartinib purchase PECy5 Ab were from Beckman Coulter (CA, USA), whereas anti-lambda PE anti-kappa allophycocyanin were from Dako (Denmark). Goat serum was purchased from Sigma-Aldrich (MO, USA). Anti-phospho-Smad1/5/8 and anti-phospho-Smad1/5 Ab, was from Cell Signaling Technology (MA, USA), anti-IRF-4 (Mum1) and anti-Actin

Ab were from Santa Cruz Biotechnology (CA, USA). Anti-XBP-1 Ab was from Abcam (UK) and Hoechst 33258 (2 μg/mL in PBS) was from Calbiochem (Germany). Peripheral blood was collected from anonymous, healthy donors at The Blood Bank in Oslo, after informed consent and with approval from regional authorities (REK S-03280). B cells were isolated using positive selection with CD19+ Dynabeads (Invitrogen) as described previously 54. IgD-depleted memory B cells were obtained by negative selection by incubating CD19+ B cells with Pan Mouse IgG Dynabeads (Invitrogen) coated with anti-mouse IgD Abs (BD) for 30 min at 4°C, followed by removal of beads. Purified B cells were cultured in X-VIVO15 (BioWhittaker, Switzerland). Proliferation

and differentiation were induced by CD40L (used at 1 μg/mL and pre-incubated with Enhancer for Ligand (1 μg/mL) for 30 min at room temperature before adding to cells) and IL-21 (50 ng/mL) in the presence or absence of rhBMP-2 (300 ng/mL), rhBMP-4 (50 ng/mL), rhBMP-6 (500 ng/mL), rhBMP-7 (400 ng/mL), mouse rNoggin (5 μg/mL) or anti-human Cytidine deaminase BMP-6 mAb (500 ng/mL). In some experiments, the number of cell divisions was tracked by labeling the cells with CFSE (Molecular Probes, OR, USA). CFSE (5 μM) in PBS was added to the cells (20×106 cells/mL) and incubated for 10 min at 37°C. The reaction was stopped by adding ice-cold PBS with 20% FCS, followed by washing and culturing of the cells as described. To measure DNA synthesis, B cells were cultured in triplicates (75 000 cells/200 μL in 96-well plates) for 3 days, and 3H-thymidine (American Radiolabeled Chemicals, MO, USA) was added for the last 16 h of incubation.

47–49 The interaction between T cells and macrophages


47–49 The interaction between T cells and macrophages

is known to be critical for prevention of bacterial growth.50–53 However, it is not clear how various M. tuberculosis proteins can trigger the Th1 response. Several factors, such as the affinity between the T-cell receptor (TCR) and peptide–MHC ligand, peptide ligand density and costimulatory signalling during T-cell activation, can play important roles selleckchem in the regulation of the Th1/Th2 T-cell response.11,12,54–57 Cytokines induced during innate activation of macrophages have also been shown to be extremely important in controlling the Th1/Th2 balance. For example, induction of IL-12 or TNF-α can trigger a Th1 response;58,59 however, if more IL-10 is produced, the response is likely to be biased towards the Th2 type response.60,61 It has been shown that various M. tuberculosis

secretory proteins bind to a specific receptor on macrophages and influence the downstream signalling cascades and the induction of pro-inflammatory cytokines.62 Although up-regulation of iNOS expression and NO production during infection with M. tuberculosis is well known, very few studies have actually identified the M. tuberculosis proteins directly involved in the up-regulation of the iNOS gene. Our study indicates that rRv2626c affects the macrophage-signalling cascades Erlotinib manufacturer and up-regulates iNOS induction and NO production mainly by increasing NF-κB activity. Interestingly, flow cytometry data indicate that Rv2626c binds to the macrophage surface with high affinity and specificity. It is possible that the specific binding of Rv2626c on the macrophage surface causes modulation of the downstream signalling pathways triggering NF-κB signalling, which results in increased induction of iNOS23 as well as the cytokines TNF-α63 and IL-12.64 Although the exact beneficial role of iNOS/NO in anti-mycobacterial Abiraterone manufacturer killing has not been uniformly elucidated,65 studies have confirmed that iNOS/NO is crucial in limiting bacterial growth.66,67 Similarly, the role of TNF-α in TB is paradoxical because, although there is evidence of its protective role,68 it can play a part in the tissue damage that

characterizes human disease.68 A recent study also indicates that M. tuberculosis activates TNF-α production to induce apoptosis of macrophages.62 Our study clearly demonstrates that the secretory M. tuberculosis Rv2626c protein induces pro-inflammatory responses by modulating the expression of iNOS and increasing the secretion of IL-12 and TNF-α, which may play an important role in the initiation of the adaptive immune response in the host. Mycobacterium tuberculosis proteins that induce the Th1 response have been used as targets for subunit vaccines. For example, use of the mycobacterial 30-kDa major secretory protein (antigen 85B, Ag85B) was found to protect animals from M. tuberculosis infection by inducing a Th1-dominant response.