Mycobacterium Tuberculosis In Macrophages Research Articles

Activities of Biapenem against Mycobacterium tuberculosis in Macrophages and Mice

ObjectiveTo assess the activities of biapenem against multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis. MethodsBiapenem/clavulanate (BP/CL) was evaluated for in vitro activity against Mycobacterium tuberculosis (Mtb) multidrug-resistant (MDR) isolates, extensively drug-resistant (XDR) isolates, and the H37RV strain. BP/CL activity against the H37Rv strain was assessed in liquid cultures, in macrophages, and in mice. ResultsBP/CL exhibited activity against MDR and XDR Mtb isolates in liquid cultures. BP/CL treatment significantly reduced the number of colony forming units (CFU) of Mtb within macrophages compared with control untreated infected macrophages. Notably, BP/CL synergized in pairwise combinations with protionamide, aminosalicylate, and capreomycin to achieve a fractional inhibitory concentration for each pairing of 0.375 in vitro. In a mouse tuberculosis infection model, the efficacy of a cocktail of levofloxacin + pyrazinamide + protionamide + aminosalicylate against Mtb increased when the cocktail was combined with BP/CL, achieving efficacy similar to that of the positive control treatment (isoniazid + rifampin + pyrazinamide) after 2 months of treatment. ConclusionBP/CL may provide a new option to clinically treat MDR tuberculosis.

Polysaccharide rich extract (PRE) from Tinospora cordifolia inhibits the intracellular survival of drug resistant strains of Mycobacterium tuberculosis in macrophages by nitric oxide induction

Plethora of clinical and scientific information obtained in recent past has strengthened the idea that targeting critical constituents of host immune system may have beneficial outcomes for the treatment of tuberculosis. Macrophages being the primary host for Mycobacterium tuberculosis, offer an attractive target for modulation. Owing to their negligible toxicity, plant derived polysaccharides with the ability to activate macrophages; are suitable candidates for immunomodulation. In the present study, effects of polysaccharide rich extract (PRE) isolated from Tinospora cordifolia, on the survival of intracellular MTB strains and activation of macrophages were investigated. PRE treatment up regulated the expression of pro-inflammatory cytokines such as IL-β, TNF-α, IL-6, IL-12, and IFN-γ in RAW 264.7 cell line. Up regulation in the expression of NOS2 was observed along with concomitant enhanced nitric oxide production post PRE treatment. Surface expression of MHC-II and CD-86 was up regulated after PRE treatment. Above results suggested the classical activation of macrophages by PRE treatment. Furthermore, PRE treatment led to the activation of all the three classes of MAPK i.e p38, ERK and JNK MAPKs. Further, PRE up regulated the expression of cytokines, NOS-2, MHC-II and CD-86 in MTB infected macrophages. PRE treatment inhibited the intracellular survival of drug resistant MTB in macrophages which was partially attributed to PRE mediated NO induction. Thus our data demonstrate classical activation of macrophages by PRE treatment and killing of intracellular MTB by NO induction.

Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we determined that this mutant assimilated 10-fold less Bodipy-palmitate than the wild type strain during infection in macrophages. This quantitative method of fatty acid uptake can be used to further identify pathways involved in lipid uptake by intracellular Mtb and possibly other bacteria.

Thymoquinone (TQ) inhibits the replication of intracellular Mycobacterium tuberculosis in macrophages and modulates nitric oxide production

BackgroundHuman tuberculosis, which is caused by the pathogen Mycobacterium tuberculosis, remains a major public health concern. Increasing drug resistance poses a threat of disease resurgence and continues to cause considerable mortality worldwide, which necessitates the development of new drugs with improved efficacy. Thymoquinone (TQ), an essential compound of Nigella sativa, was previously reported as an active anti-tuberculosis agent.MethodsIn this study, the effects of TQ on intracellular mycobacterial replication are examined in macrophages. In addition, its effect on mycobacteria-induced NO production and pro-inflammatory responses were investigated in Mycobacterium tuberculosis (MTB)-infected Type II human alveolar and human myeloid cell lines.ResultsTQ at concentrations ranging from 12.5 to 25 μg/mL and 6.25 to 12.5 μg/mL reduced intracellular M. tuberculosis H37Rv and extensively drug-resistant tuberculosis (XDR-TB) 72 h post-infection in RAW 264.7 cells. TQ treatment also produced a concentration-dependent reduction in nitric oxide production in both H37Rv and XDR-TB infected RAW 264.7 cells. Furthermore, TQ reduced the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory molecules such as tumor necrosis factor-alpha (TNF-α) and interlukin-6 (IL-6) in H37Rv-infected cells and eventually reduced pathogen-derived stress in host cells.ConclusionsTQ inhibits intracellular H37Rv and XDR-TB replication and MTB-induced production of NO and pro-inflammatory molecules. Therefore, along with its anti-inflammatory effects, TQ represents a prospective treatment option to combat Mycobacterium tuberculosis infection.

Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages.

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti-tubercular agents using a fragment-based drug design approach.

With the emergence of extensively drug-resistant tuberculosis, there is a need for new anti-tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M.tuberculosis and M.bovis BCG. We generated a mutant strain of M.bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M.bovis BCG and M.tuberculosis. The hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct 'hits' from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X-ray crystallography. The compounds also inhibited growth of M.tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol-supplemented minimal medium. We propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti-tubercular drugs. This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Curcumin enhances human macrophage control of Mycobacterium tuberculosis infection

With the worldwide emergence of highly drug-resistant tuberculosis (TB), novel agents that have direct antimycobacterial effects or that enhance host immunity are urgently needed. Curcumin is a polyphenol responsible for the bright yellow-orange colour of turmeric, a spice derived from the root of the perennial herb Curcuma longa. Curcumin is a potent inducer of apoptosis-an effector mechanism used by macrophages to kill intracellular Mycobacterium tuberculosis (MTB). An in vitro human macrophage infection model was used to determine the effects of curcumin on MTB survival. We found that curcumin enhanced the clearance of MTB in differentiated THP-1 human monocytes and in primary human alveolar macrophages. We also found that curcumin was an inducer of caspase-3-dependent apoptosis and autophagy. Curcumin mediated these anti-MTB cellular functions, in part, via inhibition of nuclear factor-kappa B (NFκB) activation. Curcumin protects against MTB infection in human macrophages. The host-protective role of curcumin against MTB in macrophages needs confirmation in an animal model; if validated, the immunomodulatory anti-TB effects of curcumin would be less prone to drug resistance development.

Vitamin D: Immuno-modulation and tuberculosis treatment.

Tuberculosis (TB) is a major global health problem and often coincides with vitamin D deficiency. High doses of vitamin D were widely used to treat TB during the pre-antibiotic era. Vitamin D exerts its action through vitamin D receptor (VDR), and VDR gene polymorphisms are associated with susceptibility or resistance to tuberculosis as well as sputum smear and culture conversion during anti-TB treatment. In-vitro studies have revealed that 1,25-dihydroxyvitamin D3 enhances innate immunity by increased expression of various antimicrobial peptides, including cathelicidin, and induction of autophagy of the infected cells thus restricts the intracellular growth of Mycobacterium tuberculosis in macrophages. On the other hand, vitamin D has been shown to suppress the pro-inflammatory cytokine response and enhance the anti-inflammatory response. Supplementation with vitamin D in concert with treatment for TB may be beneficial with respect to minimizing the excessive tissue damage that occurs during the active stage of tuberculosis disease. Several clinical trials have evaluated vitamin D supplementation as an adjunct therapy in the treatment for tuberculosis. However, results are conflicting, owing to variations in dose regimens and outcomes. Further investigations are needed to find the optimal concentration of vitamin D for supplementation with standard anti-TB drugs to optimize treatment, which could help to effectively manage both drug-sensitive and drug-resistant tuberculosis.

Evaluating the Association between TaqI Variant of Vitamin D Receptor Gene and Susceptibility to Tuberculosis: A Meta-analysis.

Objectives:Vitamin D has been shown to hamper the growth of Mycobacterium tuberculosis in macrophages. The actions of vitamin D are exerted through a vitamin D receptor (VDR). The genetic variant TaqI of VDR has been implicated in tuberculosis (TB) risk in several case-control studies. However, these studies have shown inconsistent results. Hence, a meta-analysis was conducted to investigate the potential relationship between VDR TaqI polymorphism and risk of developing TB.Materials and Methods:We performed a quantitative synthesis for published studies based upon the relationship between TaqI polymorphism and TB risk from PubMed (Medline) and Embase databases. The meta-analysis was performed and pooled odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for all genetic models.Results:A total of 21 studies including 2,960 TB cases and 3,894 controls were included in this study. The pooled analysis demonstrated no evidence of association between VDR TaqI genotypes and risk of TB in any of the genetic models; variant (t vs T: P = 0.618; OR = 1.051, 95% CI = 0.864–1.278), homozygous (tt vs TT: P = 0.120; OR = 1.336, 95% CI = 0.927–1.924), heterozygous (Tt vs TT: P = 0.925; OR = 0.988, 95% CI = 0.774–1.262), dominant model (tt + Tt vs TT: P = 0.805; OR = 1.032, 95% CI = 0.805–1.322), and recessive model (tt vs TT + Tt: P = 0.180; OR = 1.229, 95% CI = 0.909–1.660). No publication bias was detected during the analysis.Conclusions:Overall findings of this meta-analysis suggest that genetic polymorphism TaqI of VDR gene may not contribute to the risk of TB. However, future larger studies with group of populations are warranted to analyze this relationship.

Inhibition of Intracellular Survival of Multi Drug Resistant Clinical Isolates of Mycobacterium tuberculosis in Macrophages by Curcumin

Curcuma longa commonly known as turmeric has been used in Indian Ayurvedic medicine as a constituent to treat various disorders. It is by now clear that principal curcuminoid of turmeric; curcumin, a yellow pigment, is responsi- ble for these beneficiary activities. The aim of the present study was to evaluate anti-mycobacterial effect of curcumin (CMN) on intracellular growth of MDR clinical isolates of Mycobacterium tuberculosis (MTB). Curcumin was evaluated for its efficacy to inhibit the intracellular growth of MTB H37Rv and two MDR clinical isolates in Raw 264.7 cell line us- ing CFU assay. Resazurin microtiter plate assay (REMA) was used to evaluate its direct anti-mycobacterial activity. Curcumin, though did not show direct anti-mycobacterial activity against three MTB strains, exhibited dose dependent in- hibition of intracellular growth for MTB H37Rv as well as two MDR clinical isolates. These results suggest that CMN could be a potential candidate for future, novel adjunctive anti-TB therapy.

Rapid determination of growth inhibition of Mycobacterium tuberculosis by GC–MS/MS quantitation of tuberculostearic acid

Classical determination of growth inhibition of Mycobacterium tuberculosis in macrophages and mice by new candidate anti-TB drugs utilizes the determination of colony forming units (CFUs) from lung homogenates, a labor-intensive process requiring 2-3 weeks incubation. Qualitative analysis of tuberculostearic acid (TBSA), a cell wall associated biomarker found in M. tuberculosis, has been investigated for clinical diagnosis of tuberculosis (TB) but few reports exist of attempts to quantitate TBSA. Gas chromatography-mass spectroscopy (GC-MS/MS) was used in quantitating the derivatized methyl ester of TBSA during growth of M. tuberculosis in axenic medium, macrophage cultures and in the lungs of gamma interferon knockout (GKO) mice with and without exposure to anti-TB agents. The quantity of TBSA methyl ester (TBSAME) in the absence of and following exposure to anti-TB drugs was positively correlated with CFU in all three models. The stability of TBSA precludes its use as a surrogate for bactericidal activity but its exceptional thermal stability enables lung homogenates to be autoclaved prior to analysis. GC-MS/MS determination of TBSA is a rapid, sensitive and accurate means of detecting growth inhibition of any strain of M. tuberculosis in cell culture and in vivo.

Vitamin D enhances IL-1β secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1β and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

Intracellular survival of Mycobacterium tuberculosis in macrophages is modulated by phenotype of the pathogen and immune status of the host

Emerging evidence indicates that the causative agent of tuberculosis is more genetically and functionally diverse than appreciated previously. The impact of this variation on the clinical manifestation of the disease remains largely unknown. In addition, there exists significant variability in the immune status of the host governing susceptibility to tuberculosis. The effect of these variations on the host pathogen interaction was investigated by taking varying pathogen phenotypes (virulent H37Rv, a-virulent H37Ra and a multidrug resistant strain #591) and varying host (18 MDR-TB and 16 fresh TB patients and 10 healthy, BCG-vaccinated individuals). The key question was whether the intracellular survival of Mycobacterium tuberculosis (MTB) in human monocyte-derived macrophages (MDM), an attribute of pathogenic potential, can be modulated by the immune status of the hosts or phenotype of MTB.The findings of this study indicate that induction of TNF-α may not be a global indicator of virulence of a strain. TNF-α release may be differentially regulated in response to the same strain depending upon the immune status of the host. Moreover, the phenotype of the infecting MTB and the host’s immune status played a comparable role in the intracellular survival of MTB. This picture supports the hypothesis that in addition to the phenotype variation of the mycobacteria, the immune status of an individual will greatly influence the outcome of the host–pathogen interaction. These results may have a bearing on the future endeavors in vaccine development and TB control strategy.

Interactions between Naïve and Infected Macrophages Reduce Mycobacterium tuberculosis Viability

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

Divergence of immunologic and protective responses of different BCG strains in a murine model

The ongoing evolution of BCG after its introduction in 1921 resulted in strains that differ genetically and phenotypically. Based on a genomic deletion (Region of Difference 2 or RD2) that occurred between 1927 and 1931, BCG strains can be sub-classified by the presence or absence of RD2. The existence of other mutations that distinguish BCG strains precludes simple comparison of RD2-positive and RD2-negative BCG strains to determine the importance, if any, of RD2 for vaccine protection. In this study, we have compared the RD2-containing BCG Russia, BCG Pasteur (which is a natural mutant for RD2), and BCG Pasteur complemented with RD2-genes Rv1979c-Rv1982 through various in vitro and in vivo assays of immunogenicity and protection. We determined that the presence of RD2 did not affect vaccine persistence, but lead to increased immunogenicity, as measured by ELISpot. Additionally, T-cells from animals immunized with BCG Russia and BCG Pasteur::Rv1979c-82 were more effective at killing Mycobacterium tuberculosis in macrophages than T-cells from animals immunized with BCG Pasteur. In a mouse vaccine-challenge model, the presence of RD2 had no effect on pulmonary TB, as measured by M. tuberculosis burden and degree of histopathology, at 4, 8 or 12 weeks post-infection. The presence of RD2 was however associated with decreased dissemination of M. tuberculosis to the spleen. Together, our data demonstrated that the loss of RD2 resulted in decreased immunogenicity but did not affect protection against pulmonary TB, indicating a dissociation between these phenotypes associated with BCG vaccination.

Autophagy as a basis for the health-promoting effects of vitamin D

Autophagy is an evolutionarily conserved lysosomal self-digestion process essential for cellular homeostasis, differentiation and survival. As an adaptive response, it protects organisms against a wide range of pathologies, including cancer, infection, neurodegeneration, heart disease and ageing. Thus, compounds activating autophagy could have great potential in the prevention of common diseases. Interestingly, recent data link autophagy to two functions of the active form of vitamin D (VD): the induction of cancer cell death and the clearance of Mycobacterium tuberculosis in macrophages. Because VD deficiency is associated with many pathologies resembling those induced by defective autophagy, it is tempting to speculate that autophagy plays a more general role in the multiple health-promoting effects of VD.

Differential recruitment of CD63 and Rab7-interacting-lysosomal-protein to phagosomes containing Mycobacterium tuberculosis in macrophages

M.tb is an intracellular pathogen which survives within the phagosomes of host macrophages by inhibiting their fusion with lysosomes. Here, it has been demonstrated that a lysosomal glycoprotein, CD63, is recruited to the majority of M.tb phagosomes, while RILP shows limited localization. This is consistent with the author's findings that CD63, but not RILP, is recruited to the phagosomes in macrophages expressing the dominant negative form of Rab7. These results suggest that M.tb phagosomes selectively fuse with endosomes and lysosomes to escape killing activity while acquiring nutrients.

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages.

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that siderocalin expression is upregulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or overexpressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen. A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis.

Effects of omega-3 and -6 fatty acids on Mycobacterium tuberculosis in macrophages and in mice

We recently showed that treatment of macrophages prior to Mycobacterium tuberculosis infection with the pro-inflammatory omega-6 lipid, arachidonic acid (AA) enhanced bacterial killing whereas the anti-inflammatory, omega-3 lipid eicosapentaenoic acid (EPA) stimulated bacterial growth. Here we tested if these effects were depending on when lipids were added to macrophages: before or during Mycobacterium smegmatis or M. tuberculosis infection. Collectively, our data suggested that a high omega-6 diet might be beneficial against mycobacteriosis, while a high omega-3 diet might be detrimental. AA also stimulated TNF-α secretion in M. tuberculosis-infected macrophages whereas EPA inhibited this process. AA strongly activated the MAP kinase p38 in uninfected cells but M. tuberculosis infected cells blocked the ability of AA to activate p38; AA-dependent killing is therefore independent of p38. We therefore tested diets enriched in omega-3 and omega-6 lipids on a mouse model of tuberculosis. In contrast to the in vitro results, the omega-6 tended to increase survival of M. tuberculosis in mice, while omega-3- tended to increase pathogen killing. Overall our results together with those previously reported in the literature suggest that it is almost impossible to predict, at the whole organism level, if a diet enriched in omega-3 or -6 will be beneficial or detrimental to intracellular pathogens.