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Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple's disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection.
Author Summary
Innate immune cells are sentinels allowing the host to sense invading pathogens. Among them, macrophages are highly microbicidal and are able to kill microorganisms. However, several pathogens have evolved strategies to hijack macrophage responses in order to survive or replicate. Tropheryma whipplei is the agent of Whipple's disease, a systemic disease that associates arthropathy, weight loss and gastrointestinal symptoms. It has been known for several years that this bacterium has a tropism for macrophages, in which it replicates. In this study, we have shown that T. whipplei induces host cell apoptosis and a surprising macrophage activation, characterized by anti-inflammatory molecules and type I interferon (IFN) signaling, which is generally associated to viral infections. We demonstrate that this type I IFN response is critical for bacterial pathogenicity, as it is required for bacterial replication and provides the first step of the apoptotic program of infected macrophages. By identifying these signaling events induced in macrophage by T. whipplei, we can now better understand the molecular basis of the pathophysiology of Whipple's disease, of interest for clinical and therapeutic ends.
Citation: Al Moussawi K, Ghigo E, Kalinke U, Alexopoulou L, Mege J-L, Desnues B (2010) Type I Interferon Induction Is Detrimental during Infection with the Whipple's Disease Bacterium, Tropheryma whipplei. PLoS Pathog 6(1): e1000722. https://doi.org/10.1371/journal.ppat.1000722
Editor: Denise M. Monack, Stanford University School of Medicine, United States of America
Received: June 24, 2009; Accepted: December 10, 2009; Published: January 15, 2010
Copyright: © 2010 Al Moussawi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors received no specific funding for this article.
Competing interests: The authors have declared that no competing interests exist.
Over the past decades, activated macrophages were mainly considered as cells that secrete inflammatory mediators and kill intracellular pathogens. However, studies have now revealed activated macrophages as a continuum of cells with phenotypic and functional heterogeneity [1],[2]. Schematically, macrophages exposed to the classic activation signals (lipopolysaccharide (LPS) and/or IFN-γ) polarize into the M1 phenotype and express high levels of TNF, IL-1, IL-6, IL-12, type I IFN, inflammatory chemokines, such as CXCL10, and inducible nitric oxide synthase. In contrast, M2 macrophages, induced by IL-4, IL-10 or immune complexes, are characterized by the expression of non-opsonic receptors, arginase, and the absence of proinflammatory cytokines [3],[4]. Recently, we defined a "common host response" of macrophages to bacterial infections, characterized with an M1 signature and associated with the control of acute infections. However, successful infection by pathogenic intracellular bacteria usually relies on the perturbation or avoidance of the classical M1 proinflammatory activation profile [3].
Recognition of microorganisms by macrophages is mediated by pattern recognition receptors (PRR) that bind conserved microbe-associated molecular patterns (MAMPs) [5]. PRR engagement by MAMPs activates a major signaling cascade that leads ultimately to the activation of mitogen-activated protein (MAP) kinases and the transcription factors NF-κB and IRF3 [5],[6]. These transcription factors then migrate to the nucleus where they drive the transcription of proinflammatory genes and type I IFN genes, respectively [7]. Type I IFN are responsible for inducing transcription of a subset of genes referred as interferon stimulated genes. Classically, type I IFN transcription is first activated by signals that induce cooperative binding of the transcription factors c-Jun/ATF2, NF-κB and interferon regulatory factor-3 (IRF3) to the promoter [8]. Following stimulation with viral or bacterial components, the constitutively expressed IRF3 is phosphorylated in the cytoplasm, dimerizes and then translocates in the nucleus to induce the transcription of type I IFN [9]. Once secreted, type I IFN initiates a positive feed-back loop through binding to its receptor IFNAR [8]. IFNAR activates the protein tyrosine kinases JAK1 and JAK2 which phosphorylate STAT1 and STAT2 to further drive the transcription of a large group of IFN inducible genes [10].
Stimulation with Gram-negative bacteria or LPS induces type I IFN, at least partially through Toll-like receptor (TLR) 4 [11]. In addition, the intracellular pathogens Shigella flexnerii, Legionella pneumophila and Francisella tularensis induce a potent type I IFN response while non invasive mutants do not [12]–[14]. MAMPs from Gram-positive bacteria are also able to induce type I IFN. Indeed, Listeria monocytogenes triggers type I IFN, probably through bacterial DNA recognition by a cytosolic receptor [15],[16]. Infection of various cell types with Mycobacterium tuberculosis has also been shown to induce type I IFN [17]. Recently, the extracellular pathogen group B Streptococcus has been shown to induce type I IFN in a TLR-independent manner through intracellular recognition of its DNA [18]. Remarkably, stimulation of macrophages with most of these bacteria and/or bacterial ligands induces M1 polarization, strongly supporting the fact that type I IFN response is a feature of classical activation of macrophages. This point is strengthened by the fact that type I IFN significantly contribute to the cross-talk between the MyD88-dependent and MyD88-independent pathways, enabling full responsiveness to LPS [19].
Here, we have studied and characterized mouse macrophage responses to infection with the facultative intracellular Gram positive bacterium Tropheryma whipplei, the etiologic agent of Whipple's disease [20]. Whipple's disease is a rare systemic disease that associates arthropathy, weight loss and gastrointestinal symptoms [21] but its pathophysiology remains largely unknown. Recently, we provided major insights into the understanding of host immune factors in Whipple's disease, delineating macrophage polarization and apoptosis as critical in the pathophysiology of the disease [3], [22]–[24]. In this report, we provide evidence that, besides M2 macrophage polarization and apoptosis, T. whipplei induced a robust type I IFN response. This response required bacterial viability and was associated with bacterial intracellular replication. We also observed that T. whipplei induced macrophage apoptosis in a type I IFN- and JNK- dependent manner. These findings reveal an unexpected type I IFN response associated with M2 polarization.
Transcriptional program induced by T. whipplei
To evaluate gene expression profiles, bone marrow-derived macrophages (BMDM) were infected with T. whipplei for 6 h and transcriptional response was examined by microarray analysis. Of the 43,379 spotted features, 356 were significantly modulated in response to T. whipplei infection (P<0.01, Fig. 1A). To increase the reliability of our datasets, we considered transcripts as significantly regulated if they showed at least a 2-fold modulation in gene expression levels. We overall identified 59 and 11 genes that were up- and downregulated, respectively. Upregulated genes were assigned to biological process gene ontology (GO) categories. Around 50% of them belonged to the immune response GO group (Fig. 1B). These immune response genes could be sub-classified in 4 functional categories. In the first category were genes linked to macrophage polarization and more specifically to M2 polarization (Fig. 1C). Indeed, genes for the prototypal M2 markers interleukin 1 receptor antagonist (il1rn) and arginase 2 (arg2), as well as the M2 chemokines Ccl17 and Ccl22 were induced, while none of the M1 markers were modulated. The second set of the immune response-related genes represented genes related to PRR (Fig. 1C). In this group, 2 genes encoding lectins were markedly induced: clec4e which encodes a C-type lectin, and olr1, which encodes the lectin-like oxidized low density lipoprotein receptor 1. In addition, T. whipplei also upregulated the expression of tlr2, involved in the recognition of Gram-positive bacteria, and that of formyl peptide receptor 2, encoded by fpr2, which mediates the chemotactic activity of a variety of pathogen and host-derived peptides. The third group of immune response-associated genes included apoptosis-related genes. Indeed, we found that fas, and tnfsf10 were efficiently induced in BMDM following stimulation with T. whipplei (Fig. 1C). Finally, we isolated a fourth set of immune response-related genes that contained genes involved in the type I IFN response (Fig. 1C). In this group were found the genes encoding Mx1 and Mx2, which mediate resistance against negative-strand RNA viruses, but also the IFN-stimulated genes ifit1, ifit2 and ifit3, also known as isg56, isg54 and isg49, respectively. Three other IFN-inducible genes (irg1, ifi44 and gbp2) were among the most induced genes by T. whipplei. Selected genes were studied by quantitative real time RT-PCR. Upregulation of these exemplary genes was confirmed and statistical analysis revealed a significant correlation between microarray and RT-PCR data (Table 1).
BMDM were stimulated with T. whipplei (MOI 50∶1) for 6 h and transcriptional responses were monitored by microarrays. (A) Gene expression values were normalized by trimmed means followed by the unpaired Student's t test (P<0.01) and agglomerative hierarchical clustering of 356 spotted features was based on expression patterns between samples. Rows colorimetrically represent expression values of individual genes from black (low expression) to red (high expression). (B) T. whipplei-regulated genes were assigned functional categories by determining genes statistically over represented through Gene Ontology (GO) annotations. The diagram displays all statistically relevant GO groups among the up-regulated genes. (C) Immune response GO group-belonging genes were then classified in functional sub-categories after dividing gene expression values from T. whipplei-stimulated BMDM by expression values from mock-treated values to derive fold change ratios.
T. whipplei-induced M2 polarization is related to a weak proinflammatory signaling and favors bacterial replication
The transcriptional program of BMDM elicited by T. whipplei revealed a marked polarization towards a M2 phenotype. This macrophage functional activation state is characterized by the absence of proinflammatory mediators [3]. We investigated the lack of proinflammatory response by examining the activation of the transcription factor NF-κB and the phosphorylation of MAPK in response to T. whipplei.
NF-κB activation was assessed by determining changes in cytoplasmic IκBα protein levels. LPS (100 ng/ml) clearly induced NF-κB activation. Indeed, a transient degradation of IκBα, maximal at 15 min was observed (Fig. 2A). This profile was in agreement with kinetics of RelA translocation in the nucleus (Fig. 2B). In contrast, stimulation with T. whipplei induced a faint IκBα degradation between 1 h and 2 h and IκBα levels increased back normal by 3 h (Fig. 2A). However, RelA translocation was not observed, even by increasing 4 fold the dose of bacteria (Fig. 2B). Nevertheless, the fact that IκBα increased back to initial levels at 3 h suggest that T. whipplei is a weak activator of NF-κB.
(A) BMDM were stimulated with LPS (100 ng/ml) or T. whipplei (MOI 50∶1) for the indicated time points, and lysates were analyzed by immunoblotting. IκBα blots were stripped and reprobed for ERK as loading control. One representative experiment is shown (n = 3). (B) BMDM were stimulated for 1 h with LPS (100 ng/ml) or T. whipplei (MOI 50∶1 or 200∶1). RelA nuclear translocation was determined by immunofluorescence using p65-specific antibodies. (C) BMDM were stimulated with LPS (100 ng/ml) or T. whipplei (MOI 50∶1) for the indicated time points, and lysates were analyzed by immunoblotting. Phospho-p38, phospho-ERK and Phospho-JNK blots were stripped and reprobed for p38 and ERK as loading controls. One representative experiment is shown (n = 3).
Besides NF-κB, we assessed MAPK activation in response to T. whipplei. BMDM were stimulated with 100 ng/ml LPS or T. whipplei for 15 min to 3 h and, subsequently, analyzed for phosphorylation of the MAPKs, p38, Erk1/2, and JNK. In the first 15–30 min after LPS stimulation, transient phosphorylation of all kinases could be detected (Fig. 2C). In contrast, when BMDM were stimulated with T. whipplei, no phosphorylation of the MAPKs p38, ERK and JNK could be observed during the 3 hour-time-frame (Fig. 2C). Increasing the doses of T. whipplei had no effect on MAPK activation (data not shown).
As BMDM were poorly proinflammatory, it is likely that they allowed T. whipplei replication. Therefore, BMDM were infected with T. whipplei and bacterial uptake and replication was assessed by qPCR. BMDM efficiently internalized T. whipplei as around 6,000 bacterial DNA copies were detected after 4 h of infection (Fig. 3A). In the first 3 days, bacterial DNA copy number decreased and started to increase after 6 days and reached around 30,000 copies after 12 days (Fig. 3A). These results were further investigated by examining the vacuole containing T. whipplei at day 12 post infection. The great majority of bacteria colocalized with the late phagosome marker lamp1 (92%±11%); however, these T. whipplei-containing vacuoles excluded the lysosomal hydrolase cathepsin D (20%±15%, Fig. 3B).
(A) BMDM were infected with T. whipplei (MOI 50∶1) for 4 h (uptake), washed and incubated for different periods. Levels of bacterial DNA copy number were determined by qPCR (n = 3). (B) At day 12 post infection, T. whipplei organisms, lamp-1 and cathepsin D were visualized by laser scanning microscopy.
Overall, these results showed that T. whipplei infects BMDM, induces M2 polarization and replicates, at least by interfering with phagosome conversion.
Functional type I IFN signalling induced by T. whipplei
Besides M2 polarization, BMDM response profiling to T. whipplei infection revealed a striking induction of type I IFN-inducible genes. Some genes, among which ifnb1 and cxcl10, which encode respectively IFN-β and the chemokine Cxcl10, were excluded from the analysis when we applied our criterion; however, ifnb1 was up-regulated 1.6 times and cxcl10 4.2 times. To further confirm type I IFN induction following T. whipplei infection, we performed time course experiments. Expression of IFN-β mRNA increased to reach a maximal level at 6 h after infection and then was shut off, as revealed by its low expression value at 24 h (Fig. S1A). Consistent with transcriptional data, IFN-β protein was secreted by infected BMDM at 3 h and reached maximal levels 6 h post infection (Fig. S1B), exemplifying the importance of the type I IFN pathway during T. whipplei infection.
Induction of IFN-β is thought to depend on the constitutively expressed transcription factor IRF3 [9]. We therefore, determined the subcellular localization of IRF3 following T. whipplei stimulation. Raw 264.7 macrophages overexpressing EGFP-IRF3 were incubated with T. whipplei for 4 h. Confocal microscopy allowed to visualized a marked nuclear translocation of IRF3 in response to T. whipplei (Fig. 4A) while, in unstimulated cells, IRF3 remained in the cytosol. This result suggests that the type I IFN induction depends on the transcription factor IRF3. To further examine the role of IRF3 in type I IFN induction by T. whipplei, we used siRNA technology. Transfection of IRF3-specific siRNA (si-IRF3) in Raw 264.7 macrophages resulted in a dramatic reduction of IRF3 levels at 24 h (84%), as determined by Western blot, while control scramble siRNA (si-SCR) had no effect (Fig. S2A). IRF3-specific siRNA action was transient since IRF3 levels were back to normal 48 h post transfection. Therefore, we selected the 24 h time point to monitor the effect of IRF3 inhibition on IFN-β expression. Inhibition of IRF3 led to a profound reduction of IFN-β expression following T. whipplei stimulation, compared to control siRNA (Fig. 4B). As a result, IFN-β production was reduced by 94% in cells lacking IRF3 (Fig. S2B), thus identifying IRF3 as a component of the signalling pathway leading to type I IFN induction by T. whipplei-infected macrophages.
(A) RAW cells were transiently transfected with IRF3-EGFP before stimulation with T. whipplei (MOI 50∶1) for 4 h. Nuclei were stained with DAPI. (B) RAW cells were transiently transfected with IRF3-specific siRNA (si-IRF3), control scramble siRNA (si-SCR) or left untransfected (control) 24 h before stimulation with T. whipplei (MOI 50∶1). IFN-β expression was monitored after 6 h using qRT-PCR. Results are expressed as the ratio of expression levels in infected cells vs. uninfected cells relative to β actin). (C) BMDM from wild-type and MyD88/TRIF−/− mice were stimulated with T. whipplei (MOI 50∶1) for 6 h and IFN-β expression was monitored using qRT-PCR. Results are expressed as the ratio of expression levels in infected cells vs. uninfected cells relative to β actin). (D) BMDM from wild-type and IFNAR1−/− mice were stimulated for indicated time points with T. whipplei (MOI 50∶1), and lysates were analyzed by immunoblotting. Phospho-STAT1 blots were stripped and reprobed for p38 as loading control. One representative experiment is shown (n = 3). (E) BMDM from wild-type and IFNAR1−/− mice were stimulated with T. whipplei (MOI 50∶1) for 6 h and host responses were monitored using qRT-PCR on indicated genes. Results are expressed as the ratio of expression levels in infected cells vs. uninfected cells relative to β actin). (F) BMDM were stimulated with live and heat-killed T. whipplei (MOI 50∶1) for 6 h and host responses were monitored by qRT-PCR on indicated genes. Results are expressed as the ratio of expression levels in infected cells vs uninfected cells relative to beta actin.
In order to understand how T. whipplei turns on the type I IFN response, we examined the potential contribution of TLRs, the signalling of which is known to ultimately involve the adaptor molecules MyD88 and/or TRIF [25]. BMDM from double MyD88 and TRIF-deficient (MyD88/TRIF−/−) mice, which are unable to respond to TLR agonists, were stimulated with T. whipplei and IFN-β expression was monitored by qRT-PCR. Results showed that in contrast to wt BMDM, IFN-β expression was abrogated in MyD88/TRIF−/− BMDM (Fig. 4C), suggesting that TLR signalling is required for type I IFN response during T. whipplei infection.
Next, we wondered whether T. whipplei induces IFN-inducible genes via a type I IFN autocrine loop after engagement of the type I IFN receptor [26]. Thus, we first monitored the activation of the Stat1 transcription factor, one outcome of IFN secretion and type I IFN receptor engagement [27]. STAT1 activation was measured using specific antibodies targeting STAT1 phosphorylated at tyrosine 701. As shown in Figure 4D, an increase in Stat1 Tyr701 phosphorylation after T. whipplei stimulation was evidenced at 6 h with further elevation at 24 h and 48 h. In contrast, Stat1 phosphorylation was completely absent when BMDM knocked-out for the type I IFN receptor gene (IFNAR1−/−) were stimulated with T. whipplei (Fig. 4D).
Subsequently, we analyzed the irg1, ifit1, ifit2, ifit3, mx1 and mx2 gene induction following T. whipplei stimulation in IFNAR1−/− BMDM. BMDM from wt and IFNAR1−/− mice were stimulated for 6 h and RNA were subjected to qRT-PCR. As expected, T. whipplei induced a marked expression of these genes (Fig. 4E). However, the absence of type-I IFN receptor, which blocks the type I IFN autocrine induction, dramatically inhibited irg1, ifit1, ifit2, ifit3, mx1 and mx2 gene induction by T. whipplei (Fig. 4E). Finally, only live bacteria induced type I response, as heat-killed forms of T. whipplei did not induce transcription of ifnb1, irg1, ifit1, ifit2, ifit3, mx1 and mx2 (Fig. 4F).
Overall, these results indicate that type I IFN response is induced by viable T. whipplei organisms and likely involves a type I IFN autocrine loop.
Type I IFN-dependent MAPK signalling in T. whipplei infected BMDM
As members of the MAPK family are activated following the engagement of the type I IFN receptor and participate in the generation of IFN signals [28], we treated BMDM with T. whipplei and MAPK activation was followed through their phosphorylation state at 24 h and 48 h. Interestingly, we found that p38, ERK and JNK were phosphorylated at 24 h and their phosphorylation remained detectable 48 h after T. whipplei infection (Fig. 5A). In order to examine whether this late MAPK induction was attributable to type I IFN signalling, we monitored the activation of p38, ERK and JNK in IFNAR1−/− BMDM. After stimulation with T. whipplei, p38 and ERK activities were increased at 24 h and were still detectable at 48 h (Fig. 5A). Conversely, the immunoreactive band of phospho-JNK was poorly if not detected in IFNAR1−/− BMDM stimulated for 24 h and 48 h (Fig. 5A). Densitometry of the phosphorylated p38, ERK and JNK 24-h autoradiographs confirmed the differences in band intensity (Fig. 5B). These results suggest that T. whipplei induces a late MAPK signalling, which is, at least for JNK, dependent on the type I IFN receptor engagement.
(A) BMDM from wild-type and IFNAR1−/− mice were stimulated with T. whipplei (MOI 50∶1) for indicated time points, and lysates were analyzed by immunoblotting. Phospho-p38, phospho-ERK and phospho-JNK blots were stripped and reprobed for tubulin as loading controls. One representative experiment is shown (n = 3). (B) Densitometry values of the phospho-p38, phospho-ERK and phospho-JNK autoradiographs were normalized to tubulin.
Type I IFN signalling sensitizes BMDM to cell death following T. whipplei infection
Type I IFNs are known to induce apoptosis, at least in part through up-regulation of tumor necrosis factor (TNF) family proteins such as CD95 (Fas) [29]. In addition, we previously showed that T. whipplei induces apoptosis of human macrophages and that circulating apoptotic markers are increased during active Whipple's disease [22],[23]. To explore whether T. whipplei induces apoptosis of BMDM and whether type I IFN was involved, we measured BMDM apoptosis by TUNEL assay in time course experiments. We observed a gradual increase of TUNEL-positive cells that peaked at 18 h post infection, with nearly 25% of apoptotic cells (Fig. 6A and 6B). Thereafter, the number of apoptotic cells slightly decreased and remained stable at 15%, 48 h post infection (Fig. 6B). Interestingly, double-labeling of the apoptotic nuclei and T. whipplei in BMDM revealed that apoptosis was induced in infected cells, but also in cells that had not engulfed bacteria (Fig. 6A, arrow).
(A) BMDM were infected with T. whipplei (MOI 50∶1) for 4 h and incubated for the indicated times. Apoptotic cells were revealed by TUNEL reaction, while T. whipplei organisms were revealed using a T. whipplei specific antibody. Nuclei were stained with DAPI. (B) Apoptosis was quantified by examining 3 to 5 fields per condition (100 to 300 cells each). The percentage of TUNEL-positive cells was calculated as the ratio between TUNEL-positive and DAPI-stained nuclei ×100 (white bars: uninfected cells, black bars: T. whipplei-infected cells, gray bar: DNaseI-treated cells). (C) BMDM from wild-type and IFNAR1−/− mice were infected with T. whipplei (MOI 50∶1) for 4 h and incubated for 18 h. As a control, cells were exposed to UV. Apoptosis was determined as the percentage of TUNEL-positive cells. (*, P<0.05). (D) BMDM were treated or not with the JNK specific inhibitor SP600125 for 30 min. Cells were then infected with T. whipplei (MOI 50∶1) for 4 h and incubated for 18 h. Apoptosis was determined as the percentage of TUNEL-positive cells. (*, P<0.05).
In IFNAR1−/− BMDM, T. whipplei-induced apoptosis at 18 h was significantly reduced as compared to wt BMDM (Fig. 6C). This was not due to delayed apoptosis since incubating cells for longer periods did not reveal significant changes in cell death (data not shown). In addition, UV exposure of IFNAR1−/− BMDM induced cell apoptosis at a level comparable to that of UV-treated wt BMDM (Fig. 6C), ruling out the fact that the IFN receptor would have been required for apoptosis induction.
Some studies have demonstrated that JNK plays a pivotal role in the activation of the apoptotic pathways [30]. As JNK was not activated and apoptosis was significantly reduced in T. whipplei-infected IFNAR1−/− BMDM (see Fig. 5A and 6C), we wondered if JNK was required for T. whipplei-induced BMDM apoptosis. BMDM from wt mice were treated with the JNK specific inhibitor SP600125 for 30 min prior T. whipplei infection and apoptosis was measured after 18 h. We found that JNK inhibition significantly prevented T. whipplei-induced apoptosis (Fig. 6D). Taken together, these results confirm that the transcriptional proapoptotic pattern induced by T. whipplei is functional and indicate that T. whipplei-induced apoptosis is dependent on an autocrine/paracrine loop involving type I IFN, its receptor IFNAR1 which leads to JNK activation.
Type I IFN is associated with T. whipplei replication
Puzzled by these findings, we wondered if bacterial replication was linked to type I IFN signaling. Thus, we infected BMDM from IFNAR1−/− mice with T. whipplei for 4 h and assessed bacterial replication. As expected, results showed that the type I IFN receptor was not involved in bacterial uptake, as around 7,000 bacterial DNA copies were detected after 4 h infection (Fig. 7A), which were comparable to that found in wt BMDM (Fig. 3A). However, replication of T. whipplei was reduced in these BMDM: around 10,000 bacterial DNA copies were detected at day 12 (Fig. 7A, compare with Fig. 3A, 30,000 copies at day 12), suggesting that type I IFN-dependent signaling is involved in macrophage permissivity to T. whipplei. Interestingly, we found that the killing of T. whipplei in IFNAR1−/− BMDM was associated with the maturation of T. whipplei-containing phagosomes, as T. whipplei colocalized with both Lamp1 and cathepsin D (85%±14% and 97%±5%, respectively) at day 12 (Fig. 7B). Finally, we wondered if type I IFN, JNK activation and bacterial replication were related. Hence, BMDM from wt mice were treated with a JNK-specific inhibitor. Bacterial survival and the nature of T. whipplei-containing phagosome were monitored by qPCR and confocal microscopy, respectively. JNK inhibition revealed cellular toxicity beginning at day 6. However, during the first 6 days, bacterial replication was similar in untreated and SP600125-treated BMDM (Fig. S3A). In addition, most bacteria colocalized with lamp1 (83%±13%), but not with cathepsin D (5%±3%) at day 6 in both untreated and SP600125-treated BMDM (Fig. S3B). Overall, these results showed that T. whipplei-induced type I IFN response governs bacterial replication through modulation of the phagosome conversion, independently of JNK activation.
(A) BMDM from IFNAR1−/− mice were infected with T. whipplei (MOI 50∶1) for 4 h (uptake), washed and incubated for different periods. Levels of bacterial DNA copy number were determined by qPCR (n = 3). (B) At day 12 post infection, T. whipplei organisms, Lamp-1 and cathepsin D were visualized by laser scanning microscopy.
A key requirement for dissecting the complex role of macrophages during infection is to understand how microbes activate or regulate host cells. In this study, we examined and characterized host responses induced by the facultative intracellular pathogen T. whipplei.
Using microarray analysis of bone marrow-derived macrophages, we identified 59 genes that were significantly up-regulated upon infection. By bioinformatical approach, we found that most prominent GO groups covered immune response and cell communication. A closer analysis revealed that these over-represented genes could be classified in 4 functional categories. First, we found that T. whipplei induced M2 polarization of BMDM, which is consistent with the transcriptional profile of intestinal infiltrating cells, mainly comprised of macrophages, from patients with Whipple's disease [24]. Arginase, the M2 chemokines Ccl22 and Ccl17, and the IL-1 receptor antagonist were induced in macrophage following infection as it has been described in Whipple's disease lesions [24]. M2 macrophages differ from classically activated M1 macrophages in terms of receptors, cytokine/chemokine expression, and effector functions. As a result, while M1 macrophages are microbicidal and inflammatory, M2 macrophages are rather seen as immunomodulators with diminished microbicidal activities [3]. We show that T. whipplei was able to invade and replicate within BMDM in a similar fashion to that observed in human macrophages [22], suggesting that i) mouse macrophages constitute model cells to study T. whipplei – macrophage interaction and ii) T. whipplei-induced M2 polarization is a general response to this pathogen. Macrophage PRR responsible for T. whipplei recognition are still unknown. However, TLR2 and FPR2, which encodes the mouse homolog formyl peptide receptor 2 of the human G-protein-coupled formyl peptide like receptor 1 were upregulated upon T. whipplei infection. TLR2 has been shown to be overexpressed in intestinal lesions of Whipple's disease [24]. Recently, TLR2 and the intracellular receptor nucleotide-binding oligomerization domain 2 (Nod2) have been shown to cooperate in inducing the expression of FPR2 in microglial cells [31]. As FPR2 mediates the chemotactic activity of a variety of pathogen and host-derived peptides, it may actively participate in the macrophage infiltration observed in Whipple's disease lesions.
Second and more strikingly, we found that a robust type I IFN response was induced by viable T. whipplei. As compared with the plethora of reports delineating the critical role of type I IFN in host resistance to many types of viruses, only few papers report their involvement during bacterial infections [32]. Results from our study suggest that type I IFN is induced in a MyD88-/TRIF-dependent pathway and demonstrate that, comparable to classical type I IFN triggering signals [33], IRF3 signaling is activated following T. whipplei infection. Activation of the transcription factor IRF3 is likely to be dependent on TBK1. Indeed, TBK1 is required for the activation and nuclear translocation of IRF3 in mouse embryonic fibroblasts (MEF). Moreover, Tbk1−/− MEF show marked defects in type I IFNs, Cxcl10, and RANTES gene expression after infection with either Sendai or Newcastle disease viruses or after engagement of the TLR3 and TLR4 by double-stranded RNA and LPS, respectively [34]. To our knowledge, type I IFNs have never been associated with the induction of M2 polarization of macrophages and are rather seen as M1 effectors [35]. However, our results strongly support this new association as data presented here suggest a positive feedback loop involved in BMDM response to T. whipplei. First, STAT1 was phosphorylated on Y701, and this phosphorylation was absent when we used IFNAR1−/− BMDM. It has been shown that Stat1 activation is achieved by phosphorylation on Y701 that is followed by nuclear accumulation. For full transcriptional activity, Stat1 is also phosphorylated on S727 [36]. As T. whipplei induced the expression of several IFN inducible genes, it is likely that STAT was also phosphorylated on S727. Second, we found that the transcription of these IFN inducible genes was abolished when macrophages lacking the type I IFN receptor were used.
Our microarray analysis did not reveal any genes related to proinflammatory activities of macrophages, suggesting that T. whipplei is a weak inducer of inflammatory responses. Even if T. whipplei triggered a weak IκBα degradation, RelA translocation in the nucleus was not detected, despite strong translocation when cells were treated with LPS. This may be due to the lack of sensitivity of the immunofluorescence assay since IκBα, the gene of which is under the control of NF-κB is resynthetized and reached initial values by 3 h. Consistent with the weak activation properties of T. whipplei, we were not able to detect early MAPK signaling in macrophages. However, MAPKs were activated more lately, after 24 h. The kinetics of MAPKs activation suggest that p38, ERK and JNK might be activated by a secondary signal, emanating from the initial T. whipplei-macrophage interaction. Indeed, type I IFN receptor engagement for example, has been shown to induce MAPKs [37],[38]. Nevertheless, we found that only JNK phosphorylation was absent in IFNAR1−/− BMDM, while activation of p38 and ERK was similar to that observed in their wt counterparts.
Another interesting feature of the T. whipplei - macrophage interaction revealed by this study is the induction of apoptosis. Macrophage apoptosis is probably linked to bacterial replication. Indeed, cells that are able to eliminate T. whipplei such as monocytes do not undergo apoptosis [22]. These results are strengthened by the fact that circulating levels of apoptotic markers such as nucleosomes are increased in patients with active Whipple's disease [23]. Here, T. whipplei induced BMDM apoptosis with a maximal response 18 h post infection, while heat-killed bacteria were unable to induce apoptosis (data not shown). Induction of apoptosis appeared associated with i) type I IFN response and ii) JNK signaling. Apoptosis was inhibited by around 60% when IFNAR1−/− BMDM were infected with T. whipplei. In the meantime, JNK activation was abrogated in these cells. By using a JNK-specific inhibitor, we were also able to inhibit by 50% T. whipplei-induced apoptosis. Hence, we can hypothesize that T. whipplei induces type I IFN, which binds its receptor, induces JNK phosphorylation to promote macrophage apoptosis. Recently, Jeon and colleagues have shown that type I IFNs activate a JNK-specific signaling cascade involving Rac1, MEKK1, MKK4 and leading to apoptosis through filamin B [39]. Type I IFNs have also been shown to activate JNK for the induction of apoptosis in some lymphoma cells [40]. Finally, type I IFNs also activate the caspase cascade leading to apoptosis [41]. However, we cannot rule out the hypothesis that macrophage apoptosis arise from other signals. Indeed, we found that genes encoding Fas (CD95/Apo1) and Tnfsf10 (TNF-related apoptosis-inducing ligand, TRAIL/Apo2L) were both significantly induced in BMDM in response to T. whipplei. Besides TNF itself, Fas and Tnfsf10 constitute two of the three death receptor/ligand systems that are responsible for the extrinsic induction of cell death [42]. Interestingly, Fas and Tnfsf10-dependent pathways involve JNK signaling and have been implicated in immunosuppressive and immunoregulatory functions [42],[43].
Besides its role on apoptosis induction, type I IFN appeared to be involved in replication of T. whipplei. Bacterial replication was partly inhibited in IFNAR1−/− cells, as compared with wt BMDM. We also found that in wt BMDM, bacteria colocalized with Lamp1 but not with cathepsin D, as already described [44]. In contrast, in macrophage lacking the type I IFN receptor, bacteria mostly colocalized with cathepsin D. These results suggest that type I IFN can modulate, at least in part, microbial killing. Indeed, it has been shown that type I IFNs modulate vacuolar H+-ATPase-mediated acidification [45]. Interestingly, JNK activation was not required for T. whipplei replication and alteration of phagosome maturation. The role of JNK in phagosome conversion and bacterial killing is unclear as it seems to depend both on the upstream events (engaged receptor) and the pathogen itself. Indeed, it has been shown that JNK is involved in Staphylococcus aureus killing in a TLR2-dependent pathway through generation of superoxide, while its inhibition has no effect when cells are infected with E. coli [46]. From our study, two signals are emanating from the type I IFN receptor. The first involves JNK and leads to macrophage apoptosis while the second promotes alteration of phagosome maturation and bacterial replication independently of JNK. It has been shown that stimulation with type I IFN activates phosphatydilinositol-3 kinase (PI3K) and its downstream effectors [47]. As PI3K is involved in the modulation of phagosome maturation [48], it is therefore possible that PI3K activity is modulated by T. whipplei to alter its phagosome and to favour its replication. Further studies are needed to determine from where these two signals diverge.
A growing body of evidence shows that type I IFN participate in the host response to bacterial infection. However, their effects to the host can be either favorable or detrimental. For example, type I IFN response is critical in protecting the host against the extracellular pathogen group B Streptococcus [18]. In contrast, production of type I IFN during L. monocytogenes infection sensitizes macrophages to cell death [49]. Similarly, type I IFN production also appears detrimental for the host during infection with the T. whipplei-closely related M. tuberculosis [50]. M. bovis was shown to have enhanced replication rates in macrophages treated with type I IFN [51]. Our results suggest that the type I IFN induced by T. whipplei is detrimental for macrophages. Human infection with T. whipplei is a rare event despite the environmental ubiquity of the organism. Clinical features of Whipple's disease are non specific and it is clear that identifying the molecular mechanisms involved in type I IFN responses would have both clinical and therapeutic consequences.
Materials and Methods
Mice, cell culture and bacteria
BMDM from six week-old C57BL/6 and IFNAR1−/− [52] mice were isolated as described previously [53]. Double MyD88/TRIF-deficient mice were bred from MyD88−/− [54] and LPS2−/− [55] mice. Mouse RAW 264.7 macrophages (American Type Culture Collection, ATCC N° TIB-71) were grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose containing 10% FCS.
The strain Twist-Marseille of T. whipplei (CNCM I-2202) was cultured with HEL cells and purified as described previously [22]. Heat-killed T. whipplei was prepared by heating at 80°C for 1 h. All animal experiments followed the guiding principles of animal care and use defined by the Conseil Scientifique du Centre de Formation et de Recherche Experimental Médico-Chirurgical (CFREMC) and were approved by the ethics board of the university at which the experiments were performed (Faculté de Médecine de la Timone).
Statistical analysis
All experiments were performed at least three times. One representative experiment is shown. Error bars represent SD of triplicate values from a representative experiment. *, P<0.05, Mann-Whitney's U test.
Transient transfection
The eGFP-IRF3 plasmid was kindly provided by G. Querat (Marseille, France). RAW 264.7 macrophages were transfected with eGFP-IRF3 plasmid construct using Nucleofactor (Amaxa Biosystems), according to the manufacturer's recommendations.
IRF3-specific and control scramble siRNA were purchased from Santa Cruz Biotechnology. RAW 264.7 macrophages were transfected with IRF3-specific and control siRNA using Nucleofactor (Amaxa Biosystems), according to the manufacturer's recommendations.
Macrophage infection and real-time quantitative PCR (qPCR)
T. whipplei organisms (MOI 50∶1) were added to BMDM for 4 h, washed to remove free bacteria and incubated for 12 days in RPMI 1640 containing 10% FCS and 2 mM glutamine. Every 3 days, macrophages were collected and DNA was extracted using the QIAamp DNA MiniKit (Qiagen). PCR was performed using the LightCycler-FastStart DNA Master SYBR Green system (Roche), as previously described [22].
Macrophages seeded on glass coverslips were infected with T. whipplei (MOI 50∶1) for 4 h, extensively washed to discard unbound bacteria and incubated in RPMI 1640 containing 10% FCS. At different time points, BMDM were fixed in 3% paraformaldehyde and permeabilized with 0.1% Triton X-100. Immunofluorescence labeling was performed according to standard procedures [56]. Briefly, BMDM were incubated with rabbit anti-T. whipplei (1∶2,000 dilution) antibodies (Ab) for 30 min [44] and rat anti-lamp1 (1∶1,000 dilution, clone 1D4B, purchased from DSHB) or rabbit anti-cathepsin D (1∶1,000 dilution, a gift from S. Kornfeld, Washington University School of Medicine, St. Louis, Missouri). Secondary Alexa Abs were purchased from Invitrogen and used at a 1∶500 dilution. Coverslips were mounted with Mowiol and examined by laser scanning microscopy using a confocal microscope (Leica TCS SP5) with a 63X/1.32-0.6 oil objective and an electronic Zoom 2X. Optical sections of fluorescent images were collected at 0.15-µm intervals using Leica Confocal Software and processed using Adobe Photoshop V7.0.1.
For the assessment of RelA (p65) translocation in the nucleus, the same procedure was followed except that BMDM were incubated with rabbit anti-p65 (RelA) monoclonal Ab (Cell Signaling).
Cell culture supernatants were assayed for IFN-β by ELISA (R&D Systems) according to the manufacturer's instructions.
Microarray analysis
BMDM were infected with T. whipplei for 6 h (MOI 50∶1) and total RNA was extracted using the RNeasy minikit (Qiagen). The quality and the quantity of RNA preparation were assessed using the 2100 Bioanalyzer (Agilent Technologies). The 4X44k Mouse Whole Genome microarrays (Agilent Technologies) were used. Sample labeling and hybridization were performed according to the manufacturer recommendations (One-Color Microarray-Based Gene Expression Analysis). Briefly, 300 ng of total RNA and cyanine 3-labeled CTP were used to synthesize labeled cRNA using the Low RNA Input Fluorescent Amplification Kit (Agilent Technologies). Hybridizations were performed in triplicates for 17 h at 65°C using the In situ Hybridization Kit Plus (Agilent Technologies). Slides were scanned at 5 µm resolution with a G2505B DNA microarray scanner (Agilent Technologies). Image analysis and intra-array signal correction were performed using Agilent Feature Extractor Software 184.108.40.206. Global normalization by trimmed means was applied on raw datasets using Excel (Microsoft). Discrimination between samples was performed using the unpaired Student's t test. We only considered a gene as differentially expressed if the P value from Student's t test was below 0.01 and its absolute fold change was over 2.
To identify functional categories of genes that were over-represented in the data sets of modulated genes, we assigned Gene Ontology (GO) annotation by using the freely available online tools FatiGO Search (http://babelomics.bioinfo.cipf.es/) and DAVID Bioinformatics Resources 2008 (http://david.abcc.ncifcrf.gov/).
All transcriptional profile files have been submitted to the GEO database at NCBI (accession number GSE16180).
Quantitative real-time RT-PCR
cDNA was synthesized from 1 µg of total RNA using SuperScript II RNase H reverse transcriptase (Invitrogen). Specific primers for each gene were designed using Primer3Plus, available online at http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi. The sequences of the targeted genes are listed in Table 2. Quantitative RT-PCR was performed using LightCycler-Fastart DNA Master SYBR Green (Roche Diagnostics) and data acquired with the ABI PRISM 7900 HT (Applied Biosystems). Gene expression was normalized to the β-actin gene, relative expression of respective genes was calculated using comparative threshold cycle method [57].
Western blotting
Macrophages were stimulated with either T. whipplei (MOI 50∶1) or Escherichia coli 055∶B5 LPS (100 ng/ml, Sigma). At designated times, BMDM were washed with ice-cold PBS. Cells were then scrapped in ice-cold RIPA buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1% Triton-X100, pH 7.5) containing protease inhibitor (Complete, Roche) and phosphatase inhibitor (Phosphostop, Roche) cocktails. The cell lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4°C and stored at −80°C. Cell lysates were examined for equal amounts of protein by the Bradford method using γ globulin as a standard [58]. Samples were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels, electrophoresed and transferred onto nitrocellulose membranes (Amersham). The membranes were blocked in PBS with 0.05% Tween 20 (PBST) supplemented with 3% powdered milk and then incubated with primary Abs against phospho-p38, total p38, phospho-ERK1/2, total ERK1/2, phospho-JNK, α-tubulin (Cell signaling), IκBα (Calbiochem) or IRF3 Ab (Santa Cruz) as indicated by manufacturers. The blots were washed with PBST and incubated with a secondary Ab, either horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin (Pierce) in PBST plus 3% powdered milk. The bound Abs were detected using Immobilon Western Chemiluminescent HRP substrate (Millipore).
Apoptosis determination by Terminal transferase deoxytidyl uridine end labeling (TUNEL) staining
Detection of apoptosis by TUNEL was performed using In Situ Cell Death Detection Kit, TMR red (Roche) according to the manufacturer's instructions. JNK inhibition was performed using SP600125 (Sigma) at 50 µM for 30 min prior infection. As a control, apoptosis was induced by exposing cells to ultraviolet (UV) as described previously [59]. After treatment as indicated, cells on glass coverslips were fixed in 4% paraformaldehyde for 15 min, washed in PBS and permeabilized with 0.1% Triton-X100 in 0.1% sodium citrate for 2 min. Cells were then incubated with the TUNEL mixture containing TMR-dUTP and terminal deoxynucleotidyl transferase for 1 h. Cells were washed in PBS and nuclei were stained with DAPI before mounting with Mowiol. Positive controls were carried out by incubating cells with 3 U/ml DNase I prior labeling procedures. Negative controls were done by incubating cells with label solution (without terminal deoxynucleotidyl transferase). Apoptosis was quantified as follows. Coverslips were examined in fluorescence mode with a Leica microscope equipped with a Nikon digital camera using a 10X objective lens. Three to five fields per condition (100 to 300 cells each) were observed. The number of TUNEL-positive and DAPI-stained nuclei were determined and the apoptosis percentage was expressed as the ratio between TUNEL-positive and DAPI-stained nuclei ×100.
List of LocusLink accession numbers for genes and proteins mentioned in the text
arg2, 11847; ccl17, 20295; ccl22, 20299; clec4e, 56619; cxcl10, 15945; fas, 14102; fpr2, 14289; gbp2, 14469; ifi44, 99899; ifit1, 15957; ifit2, 15958; ifit3, 15959; ifnar1, 15975; ifnb1, 15977; il1rn, 16181; irf3, 54131; irg1, 16365; mx1, 17857; mx2, 17858; myd88, 17874; olr1, 108078; tlr2, 24088; tnfsf10, 22035; trif (ticam1), 106759.
Supporting Information
Figure S1.
Time course of IFN-β expression and production in BMDM. (A) BMDM were stimulated with T. whipplei (MOI 50∶1) for the indicated time points and IFN-β expression was monitored using qRT-PCR. Results are expressed as the ratio of expression levels in stimulated cells vs. uninfected cells relative to β actin. (B) BMDM were stimulated with T. whipplei (MOI 50∶1) for the indicated time points and cell supernatants were assessed for IFN-β by ELISA (n = 3).
Figure S2.
IRF3 knock-down efficiency. (A) RAW cells were transiently transfected with IRF3-specific siRNA (si-IRF3), control scramble siRNA (si-SCR) or left untransfected (control). After 24 h and 48 h, lysates were analyzed by immunoblotting. IRF3 blots were stripped and reprobed for tubulin as loading controls. Densitometry values of the IRF3 autoradiographs were normalized to tubulin. (B) RAW cells were transiently transfected with IRF3-specific siRNA (si-IRF3), control scramble siRNA (si-SCR) or left untransfected (control) 24 h before stimulation with T. whipplei (MOI 50∶1). After 6 h, cell supernatants were harvested and IFN-β production was assessed by ELISA (n = 3).
Figure S3.
Bacterial replication proceeds independently of JNK activation. (A) BMDM were treated or not with the JNK specific inhibitor SP600125 for 30 min. Cells were then infected with T. whipplei (MOI 50∶1) for 4 h. SP600125 was added during the infection procedure. Levels of bacterial DNA copy number were determined by qPCR (n = 3). (B) At day 6 post infection and in the presence of SP600125, T. whipplei organisms, lamp-1 and cathepsin D were visualized by laser scanning microscopy.
We thank Lionel Pretat for assistance on confocal microscopy, Didier Raoult for technical assistance and helpful suggestions and Christian Capo for important intellectual input. We thank Bruce Beutler for LPS2−/− mice and Shizuo Akira for MyD88−/− mice. BD is grateful to Christelle and Ilona for stimulating discussions.
Author Contributions
Conceived and designed the experiments: BD. Performed the experiments: KAM EG. Analyzed the data: EG JLM BD. Contributed reagents/materials/analysis tools: EG UK LA. Wrote the paper: BD. Provided important intellectual input: JLM.
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Time is money! On this final episode of this four part series, How To Get Success When Starting Over in Life, the Do Over guy shares how to get more done while freeing up your time to do more of what you do best, or even better… to do more of what you WANT to do.
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Matt: Hey, this is Matt, "The Do Over Guy," and this is Your Do Over: Episode 42.
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Matt: Welcome to Your Do Over, coming to you live from downtown Los Angeles. This is the place where I show people who want more out of life, people dissatisfied with their current situation, how to start over and begin a new life, setting goals and objectives so they can create wealth and live life to the fullest.
You can jumpstart your Do Over and lay a solid foundation for a better life by downloading the Three Pillars of Creating the Ultimate Do Over for free at FreeDoOver.com. It's a 55-minute mp3 audio program that I made just for you with three specific steps on how to get success as you start over.
The Three Pillars of Creating the Ultimate Do Over will put the legs under your table. They will lay the foundation for you to achieve. They will act as your traveling success coach, and they're yours for free at FreeDoOver.com.
Okay. So I'm very excited for today as we conclude our series on how to get success when starting over in life. This is part 4. I've really been looking for ward to this episode as I wish I had embraced this concept so much sooner but before I do just want to say thank you to a few people. AJ investor left a comment on iTunes both inspirational and transformational. Thanks.
Five stars. Great stuff. I'm hooked and can't get enough of your podcast both your Do Over and your Investment podcasts are amazing. Your approach is honest, action-driven, inspirational, and transformational. Not only do you teach more effective ways of thinking and working but you also inspire your listeners into becoming better people and doing the right actions so everyone wins. Thank you for what you have done and continue doing. I look forward to both listening and doing.
AJ Investor, you are very welcome and thank you for leaving that great comment.
Another one from PD6866. Great, great, great stuff. 5 stars. Motivational, inspiring, and full of valuable information. After you start listening, you'll be hooked and you won't want to miss any of it. Thanks Matt for the great work.
PD6866, I wish I knew your names so I could thank you properly but thank you for leaving that great review. Last one, Awesomeness. 5-star rating by Wanbly. I was, am going through a rough time and this podcast has been helping me to get to the next step of my life and have a better foundation. Thanks for always making new podcast and consistently motivating me.
Wanbly, you're very, very welcome. Thank you.
Okay. Today's episode "How To Get Success When Starting in Life – Part Four." We're going to talk about getting success by leveraging your time. I mean this subject has got a lot of coverage over the last few years especially since the release of the "4-Hour Work Week."
The book written by Timothy Ferris. I mean that subject being outsourcing. For the last couple of years, I've been using a couple of websites to hire people to complete some of the more mundane tasks I have to do on a regular basis.
As time has passed, I've outsourced bigger and bigger and bigger projects. I've had some great experiences and I've had some terrible experiences. Through those experiences, I've created a system of which I leverage my time throughout outsourcing.
I get a whole lot more done and I get it done very, very inexpensively. So at first when I got started at anytime a small, little, minute, mundane task came up that had to be completed. I didn't want to do it. I would then stop my day. I'd log on to a number of virtual assistance sites and pose some jobs and wait for bids to come in.
Now if you don't know what I'm talking about, they're a bunch of online resources, websites. ELance is probably the most notable, the most popular, and the most famous. They'll give you access to something called virtual assistance. They're essentially your assistance but they work virtually. They don't have to physically be there.
I mean as long as that virtual assistance, personal presence is not required. I mean just about any tasks that you have or can think of. It can be delegated to someone else and free up your time so you can do it what you do best or you can do what you want to do. Rather than having to do what you have to do.
Okay? Some of the tasks that virtual assistance can carry out for you. They can take on your inbound calls or they can make your outbound calls. They can manage a help desk for you. They can provide email support services for your business or chat support services for your business.
They can do say Spanish telemarketing for your services if you have a Spanish client bout you don't speak Spanish yourself. You can hire someone to do that for you. They can do data entry, data conversion, data processing, and all kinds of transcription services and translation services.
Maybe they'll do your billing or your bookkeeping. They might do some coding for your website or your online presence. Maybe you need writing done for you. They need to write a brochure or they need a design a brochure. Maybe they'll conduct your account payable services or accounts receivable services. They can make your collection calls for you.
I know I used to own a record label. I used to hate to call the collector for records and stuff that I sold so I would have loved to had them back there to make those calls for me. Maybe you have a need for payroll services. A virtual assistant can do that for you. Or software development even or website design.
Maybe photo restoration or conversion of raw images or image enhancement services. They add up auditing, dashboard creation services for your website, reporting and analysis services. I mean you name it. They can do just about anything.
So as I was saying at first I would just pose jobs randomly as these types of tasks arose. It worked amazingly well with the majority of the time. Then I got to a point where I noticed I had been spending a great deal of time posting jobs and hiring people and managing these people, which was taking up basically the same amount of time if I just done the job myself.
Now at this point although this hadn't become much of a time saver, I was still in love with paying a few bucks for someone else to do all of the work for me. I guess I felt a little bit of power or significance with the whole process. I love talking amongst friends about how I had someone on the other side of the globe doing my work while I slept.
But after a while, you know, the novelty wore off, as I didn't really free up any time, as the management of these jobs was a full time gig. I mean my whole intention was to replace myself not to create a new job for myself.
Then one day, I was listening to a podcast on time management and efficiency. He had a really brilliant idea. Forget what their actual context was but I took that idea of creating a procedure list for various parts of my life.
I put one together for my very own procedures. The stuff that I do on the daily basis. I extracted those tasks from those procedures that I didn't want to do. The tasks that I didn't want to do.
I hired a virtual assistant on either a part-time or full-time basis to do them for me. I mean depending on the responsibility to carry out those responsibilities on a weekly basis. Or sometimes even a daily basis for me, I hire someone to take those on for me.
Since doing it this way, I've alleviated a bunch of time for essentially pennies and freed up a whole lot more of my time to spare it where I want or where I most valuable inside my business.
You can do this with your personal life as well but typically I'm using it for my business. I'll give you an example. With this podcast, I mean if I were to execute this procedure flawlessly of which I think, I don't know. I've only done once by myself in the last year.
I'd begin by outlining the subject matter of the episode. I would then record it and then I'd have it transcribed then I'd write a blogpost about the content then conduct search engine optimization by writing multiple articles from that blog post.
Then I'd submit those articles to article directories then I'd post the article to social sites like FaceBook and Twitter. Then I'd build links around the Internet pointing to that blogpost to increase my Google search results.
That would take me 2 to 3 days per episode then I was essentially timed to do it all over again. It was exhausting. It was a full-time gig just making this podcast and marketing around the Internet.
Not only it was exhausting, it really produced minimal results. I mean it would take essentially, I don't know, a year or two of doing that as a one-man band to experience any sort of real success on life before you'd start really seeing the results.
So what I did is I created a very detailed procedure of that entire process in a written step-by-step format then I hired a few virtual assistants to execute the procedure for me. So now, all I do is create an outline for the show.
I record the show then I post it to my podcast host. So I hired a VA that I had her subscribed to the show. So each time I post a new episode, she receives it then transcribes it. She then writes a few articles for me based off of the content then she sends them to my Search Engine Optimization expert of which then submits those articles to online directories. Then he creates the links to those articles to my blog post and to the podcast.
I mean that happens automatically and I've done something very similar with my real estate investing business in a way that all I have to do is call motivated sellers and put deals together. Everything else is essentially done for me.
I mean that type of time leverage is how to get success in a very efficient and much more enjoyable manner. Now direct you to where I've found to be the best virtual sites and believe me I've tried a bunch of them.
I know the ins and outs in the courts of each site but first I want to walk you through creating your procedure list or lists. If you've never done this before, it might be a very fun experiment to see what you actually do.
I've had a few jobs in my life for they had this procedures list where the procedure manual. It's this big, giant, thick manual that I don't think anybody ever really read. But now I've really understood the value. I understand the type of impact that can have on your efficiency, on your productivity inside your business, or even your life fall together.
Okay? Procedures. Procedures are instructions. So put yourself in the user's shoes or the person who's actually going to be carrying out the procedure and write from their perspective.
In other words, unlike other types of documentation, you don't need to give the reader very much background details. So do the following, you want to do the following. You want to write in the present tense. The user is performing the task now. Don't write in the past conditional or future tenses unless you have good reason to do so.
Then you're going to want to avoid ambiguities. You want to be really clear. You want to be concise. You want to use short words. I mean this isn't a romantic novel you're writing. There's no extra credit for creative writing.
Keep the words short and get to the point. Then you want to move from one step to the next in a very logical manner. Steps should follow each other in a very logical order. Then you want to highlight exceptions. Use a symbol to flag this as an exception on how to handle it then highlight warnings.
Again, warn the user that the caution must be used in this scenario. Warnings must stand out. Use a larger fonts or a warning icon. Then you'll going to want to reduce the word count where possible without altering the meaning of the text. You just want to keep this clear and concise in a very logical step-by-step process.
Now things not to do. Do not introduce acronyms without explaining what they mean. What does OLA mean to you? I know but most folks don't. Another thing you don't want to do. Don't be vague. Don't use the word "may" if possible as it implies the user can do something under certain conditions. Instead, be positive and tell them exactly what to do.
Okay? And don't get the sequence wrong. Make sure you get the sequence right. Steps have to be in correct order. Do not list steps that should be numbered. What I mean is that some items can be listed, for example a list of ingredients in cooking but you need to number the steps in the correct order so the cook can prepare the dish. You get the right result. I mean the best approach to writing a standard operating procedure is to perform procedure, write it, and test it then write it again.
So perform the procedure and make note of each action you take. Then test it then write it again. Number each step in the procedure. Now, every procedure lists the actions that the reader must take.
Now once you've completed your first list. First, highlight all of that which you don't want to do. Okay? Highlight the steps that you don't want to do. Second, highlight the step that you're really not very good at. Okay?
Then third highlight that of which doesn't really directly accomplish the goal. In other words, if you have a job that includes sales. You're going to highlight pretty much everything other than the items that actually make you money that actually convert the sale.
And you've got a few options. You can search for team of people or you can search for one person that can do everything for you. You'll have some tasks that are part time responsibilities. You'll have some that are full time type responsibilities.
Now the two sites of services that I like to use are LeverageMyTime.com, LeverageMyTime.com and VAOneStop.com. VAOneStop.com.
Now here's the difference between the two. I use LeverageMyTime.com for those part time tasks or those specialty tasks. The reason that I like this service is that while you're VA is working. The service actually take screenshots of their computer while you're being billed so you can be sure that when you are being billed that the VA is actually doing your work. I love this feature.
Okay? The whole site is very user-friendly as well so you go there and you'll post your job. So for example, every Monday, I'll send your report and I'll need an Excel spreadsheet created that looks like X and calculates like Y.
When it is complete, I want you to email to [email protected]. That might be a job that you hire somebody part time so every Monday, they're going to carry this certain procedure for you. You're going to send them report by Monday morning. They're going to convert it to an Excel spreadsheet that looks like the example you provided when they're done with it. They'll go email it to whether it's one of your customers, whether it's a fellow associate, or whether it's your boss.
I call your boss [email protected]. All right? Whatever it may be. That could be very much a tasks that takes you hours of which causes you to go home late on Monday nights and miss your favorite TV show or make you miss time with the family or maybe you can spend that extra time doing what you do best or what you like to do.
So how this could look for example, what I've actually done recently in my real estate investing business is that I have one Virtual Assistant post a classified ad on Craigslist, eBay Classifieds, and Back Page. I have them do that every single morning 5 days a week, Monday through Friday.
I mean that is a very mundane time consuming process that is really like freed up my mornings so I can spend more time in the gym so I can spend more time getting in shape. It's something that's really important to me. I mean those ads that they place; they drive traffic to a special landing page.
When those visitors fill out a form on that page, it sends in email to another Virtual Assistant of which calls them within 12 hours. She asks them 3 specific questions of which I gave her to ask. Based off of those answers, she will either input into a database for me with notes on the conversation or if it's really a hot lead, she will either 3-way me into the call or send me a text message with that leads information depending on the time of the day.
I've set up all those parameters in my procedure list. By setting up a system of which the same person does the same tasks everyday, I mean real efficiency can be injected into your life.
We all know that time is money. Right? For that type of virtual assistance, I recommend LeverageMyTime.com. LeverageMyTime.com. Now the other type of virtual assistant is one you might want as a full-time assistant to work for you.
Someone that going to work for you day in and day out doing certain tasks or conducting certain responsibilities that has to be conducted on a daily basis. For that type of VA or Virtual Assistant, I'll call him Vas from this point forward.
I would recommend the service VAOneStop.com. VAOneStop.com. It's a one-time fee around I don't know $300 or so. They find the exact type of VA with all of the special skills required for your job.
They'll go out and they'll search. They'll conduct interviews of I don't know maybe 10 candidates and narrow it down to the three. They'll send you those 3 Vas' information and then you can interview those 3 to see if they're right for your job.
For example, I used this service to find my SEO or my Search Engine Optimization guy. His name is Allan. I use the service to find my transcriptionist, Toni. I have them working together to keep my blogs full of content and to make sure that when certain keywords are typed into Google that I come up amongst the top results.
Now both of these services, I mean they freed up so much of my time that I get to spend the majority of my time doing what I do best and that's creating content. That's creating this podcast. That's doing deals on my real estate not shuffling papers, not building links and websites. That's actually doing the deals in my real estate investing business.
The other thing I get to do is I get to be there for my newly born son of which just turned 4 months by the way. But I get to be there without a whole lot of stress. Then you'll get to do with your time whatever you want.
Just imagine what else could you do with your time? If all that stuff that you have to do on a daily basis, the stuff that you're tired of doing, the stuff you don't like doing, the stuff that you're not very good at, the stuff that just kind of removes the fun out of work.
What if you could eliminate all of that from your day-to-day activities? What could you do with all that free time? I don't know. You know your life much better than I do. I'm sure you've already got some ideas.
Okay. So here are some tips for finding a good VA. Tip number 1: first the price. Okay? Vas can range from $4 per hour to $40 per hour. Yep. You can get much of the stuff on your procedure list completed for $4 an hour. Okay? You get a little excited already?
I know I was when I discovered that, however, I will never actually hire based on price alone. I used to do that but I won't do it anymore. So take your time to read your VA's profile, their work history, qualifications, and customer ratings.
You'll get access through those two websites that I gave you. Every once in a while I've gotten amazing work for cheap but for the most part even around the world across cultural lines, you do get what you paid for. So don't hire based on price alone.
Two, prepare for cultural differences. I mean if you're outsourcing abroad, you definitely need to be aware of the cultural differences that will arise. I mean outsourcing your calls, for example, would mean you're provider would need to be attuned to the cultural and social customs of your business.
If you're outsourcing web design, you'll going to want to make sure that the language your provider will be using fits your demographic. Customers are very observant and they can tell very quickly if the message they're reading is a low quality or it's poorly translated.
Third, location. VAs span the globe and are available on every continent. Now being an American, I found that the greatest success to experience to price ratio with Vas is in the Philippines.
They speak English better than most other countries. I found the thought process and logic to be the closest to the American culture. I don't mind paying a little extra in the Philippines for stress-free experience.
Now they are on an opposite clock as we are so if you need someone to be working during your hours that might not be the best solution but I have to find it pretty much over all the best results, the best experience.
My relationship with my VAs is so much stronger than in the Philippines than it has been anywhere else. I'm not saying there's not other good VAs. There are good VAs everywhere. It's the best success to experience to price ratio that I've experience.
Four: take one project at a time. Don't move so fast. Okay? Don't sign any long-term contracts that until you seen that this provider has had success on smaller projects. If possible, test the outsourcer first. By giving them a comparatively small task to accomplish, their ability to handle this project is going to prove whether they are a good match for the long run.
Just like the people that you interact on the day to day basis, face to face, it's important even with your VAs to know whether you click or not, whether you enjoy working with that person. It's going to make a big difference in your whole experience.
Number 5: brace yourself for challenges. Now as greater as it would be to immediately hit the ground running with your new provider, with your new VA. Realistically it's going to take some time to synchronize operations and build a professional relationship.
Now in order for outsourcing to work, you and your staff are going to need to be patient. You're going to need to be prepared for small missteps that may happen in the beginning. For example, if your provider is offshore then there's going to be this time zone that I mentioned earlier. That Matt can really hamper proper communication and setting up a realistic schedule that meets your needs.
That might take some practice and might take some adjustments and some refinements before you get it just right. If all parties are invested though a little practice and management of expectations are going to sort everything out.
Six: be aware of the full costs. Okay? Many companies outsource to save money. It's understandable but outsourcing can get pricey too. I mean are your outsourcing projects going to require you to employ telecommuters or freelance staff who work remotely?
If so, I mean, it could be costly depending on where the workers are based. What about equipment? Do the outsourcers have their own or are you funding the bill on that? Make sure to get as much information as possible about the total cost before it's too late.
Seven: are your expectations the same? Are all involved parties clear about what the end result should be? I mean miscommunication, benchmarks, milestones can cost a real, I don't know, can cost issues.
But be clear about communicating what success looks like as well when you expect to get there. I mean if possible provide the outsourcers with the concrete example of what you would like their finish products look alike.
I mean for instance perhaps you are outsourcing the redesign of your site. Is the vendor up to speed with the latest content management system and development software? Are they up to speed with the latest technology?
You should know what capabilities you want your site to have and assure that your providers has already been able to provide that level of quality to other clients before giving them the job.
Eight: get clear. I think I somewhat mentioned this twice but it's really important. Get clear. Set clear deadlines and project descriptions. One of the best buyer products of outsourcing is it teaches you how to clearly define the project.
It teaches you how to create a procedure list that standard operating procedure. It will also identify steps that often get missed when we do our own work. Without a clear project go outsourcing, I mean, it's futile.
Alright? Also properly defining a job at the beginning gives it a better chance of success. Then when you put time parameters on it, it's all that much more efficient.
Okay. That's how to get success when starting over in life by leveraging your time. My two favorite websites in fact they are the only ones that I use now. LeverageMyTime.com and VAOneStop.com.
A couple of others that I found to be really fun is fiverr.com. It's a great little place to go for people where people share things and they're willing to do for $5.
Okay? If you got small step that's not, you know, critical to the mission that might be a good place to check out. It's fiver, f-i-v-e-rr. There are 2 Rs. .com .
Then a new one that I just found called TaskRabbit.com. TaskRabbit.com. You can get just anything done, you know, safe, reliable, awesome people. Right now they're just currently serving Boston, San Francisco Bay, New York city, Chicago, Los Angeles, and Orange County inside of California.
They'll actually do stuff face-to-face. They still do stuff. They could do the stuff around the house or do stuff that you need an actual physical being in there for. Check that out. That's TaskRabbit.com.
Hey but for serious day-to-day responsibilities, go to LeverageMyTime.com and VAOneStop.com. That's it for today. God loves and so do I.
I am Matt, the Do Over guy. I will see you on the next episode of your Do Over.
Announcer: Thank you for tuning in to Your Do Over, where the ignored, underestimated, and unknown steps to producing results and making life work are revealed. And remember, knowledge is potential power. Take action on what you learned today. This is not your learn over. It's Your Do Over.
To view the resources referenced in today's show and to retrieve a complete show transcript, visit www.TheDoOverGuy.com. Stay connected with Matt, the Do Over Guy, Theriault on Twitter at TheDoOverGuy and on Facebook at www.facebook.com/dooverguru.

subset: commoncrawl


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I started September with some Christmas projects. I always wanted a white tree full of crocheted ornaments but never did something about it until now. Thank you, Joanna, from Wishes in the Rain for providing the patterns and tutorials. Aaand, I can't get We Belong by Pat Benatar out of my head. Thanks a lot, … Continue reading Hello, September
Tag: ramblings
I have so many things to do. Among many others, a few projects to finish… I also need to declutter a few areas of my house because we are going to move in a few months. But it's like my brain and my hands froze. I can't focus on anything. Anything! So many things to … Continue reading Procrastinating


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PHAQs (Philosophically Asked Questions)
A: Of course, one thing that philosophers do is to teach others what philosophy is, and how to pursue it. But that is not what you want to know.
Philosophers try to answer certain questions, or solve certain kinds of puzzles. Both the questions and puzzles, and the proposed answers to them, have certain unique characteristics.
Philosophers' questions expose unnoticed assumptions that may otherwise hold unwarranted sway over us; the questions philosophers ask are about fundamental matters having far-reaching impact across our systems of thought. They are in that respect very general. Philosophers' answers are intended to be clear and precise, and governed by very strict rules for reasoned inquiry.
Though a philosopher may share with a physicist an interest in the structure of the universe, or share with a psychologist an interest in the mind, philosophical questions are distinct from those asked within any other discipline or practice.
Q: What kind of questions or puzzles do you inquire about?
A: Perhaps an example or two from the various areas of philosophy will help.
Physics employs the concepts of space, time and causation. Philosophers wonder what these terms mean, how are they to be understood. Newton, for example, suggested that in his conception of time, it flowed like a river. Philosophers note that if time flows, it must flow at a certain rate, and that any rate of flow is a measure of change per unit of time. So Newton's account, instead of explaining time, required the prior understanding of the idea. Philosophers try to dig deeper.
The law requires that for a person to be guilty of any crime they must show the requisite intent, they must have done what they did on purpose. In legal jargon this is called mens rea. Philosophers examine the fundamental issue of what is required for an action to BE one's own, to be a product of your will.
These two examples, the puzzle about time, and freedom of the will, are topics in metaphysics, a main branch of philosophy. Both are fertile ground for philosophical inquiry.
One more example: Moralists urge certain actions as right, and others as wrong. Philosophers wonder about the nature of right and wrong acts: what is it about them that makes them so? Is an action wrong simply because it causes harm? But some harmful acts are surely correct things to do: say action taken in self-defence. Are actions right because they are an instance of some general duty we have? But what are these general duties, and what justifies these rather than some others?
A: In all of the above examples there is a general requirement to get at the truth, to say what is true. But what is truth? A first philosophical pass might be that to tell the truth is to utter a sentence which tells it like it is -- that is, the truth is a kind of correspondence between what one says and the way things are. But many have argued that one cannot make out how things are without first having a concept of truth and falsity. Theories of truth, then, are another philosophical arena.
But we can say at least this: theories and arguments which tend to track the truth (however that may ultimately be defined) are to be prized.
Q: Why so much emphasis on theories and arguments?
A: An argument cannot tell you what is true, but it can do two things of almost equal importance: it can tell you what certain given truths imply, what other claims follow from them, and they can preserve truth, insuring by correct argument that hard won truths are not thrown away by faulty reasoning. So the study of argument itself -- both formally and informally -- is another rich philosophical vein.
Q: But isn't what is right or wrong, what is unjustified or unjustified, purely a matter of opinion? Isn't my philosophy, whatever it is, just as valid as anyone else's?
A: Most philosophers would find this a curious claim to make. First note that no one is prepared to make such claims in other disciplines. No one seriously entertains the idea that anyone's opinion about physics or chemistry is just as valid as a scientist's opinions. You may have a view about the chemical composition of water, but unless it is one which agrees with the relevant experts (who say that water is H2O) then your view is inadequate.
Philosophical views are much more general than those in other disciplines, however, and seem to allow for a great deal more speculation. But this does not imply that they are without stringent criteria of acceptability. These criteria include the justification of one's central claims either by deduction from mutually acceptable, and more basic, premises, or by arguing that the proposed theory best explains the set of facts in question (with what is to count as the best explanation itself a philosophical question of some merit).
Any alternative view one might claim to hold will, equally, be asked to show its own justification from more basic and mutually acceptable premises, or how it provides a superior account of the facts that we are given.
To assert that a particular philosophical view is just the opinion of the philosopher who propounded it is to fail to do justice to the account such a philosophical view entails – its scope and its credentials.
Q: In my philosophy class, the professor explained the views of a philosopher who lived a long time ago, and then argued that they are mostly false. What's the point of that?
A: Much of the study of philosophy (and philosophy classes) is the exposition and dissection of the views of prominent philosophers, including, perhaps, your instructors. The great philosophers are just those who have seen the deepest into the various perplexities noted above, and whose views on these subjects have stood the test of time. Their views (including their mistakes) serve to articulate the problems, and orient us to their potential solution.
No philosopher can satisfactorily defend her or his views without making clear the strongest arguments against these views, and in doing so, demonstrating, by argument, how her view is successful where others are not. It is this brutally honest self-appraisal which is philosophy's salient virtue and, for some, one of its most attractive features. Thus you will often find that your instructor presents his own views and then examines arguments against them as well as doing this for other philosophers.
In short, any successful philosophy avoids bias by contending honestly with its strongest opponents.
Q: There is a lot of emphasis in the above answers on clarity and meaning, and in making sense. Why?
A: We cannot make much headway in solving any perplexity if we don't first come to some agreement on what we mean. Here is a simple example. One might wonder whether a tree falling in the forest where no one is present, makes a noise. This supposed philosophical puzzle is easily answered – we need only decide on what we mean by makes a noise. If we mean produces compression waves (sound waves) in the surrounding medium, then surely it does make a noise. If we mean, produces an auditory sensation in a listener's mind, then, by hypothesis, it does not make a noise.
This puzzle is, of course, a simple one. But other concepts which play fundamental roles in our metaphysical, ethical, and political concerns are not nearly so happily resolved. What counts as a person? What is death? What, if anything, is a soul? What makes an act just? These, and many more (including, as we have already noted, the concepts of space and time, right and wrong) are only a few of the yet to be completely resolved philosophical problems. Finding an account for these concepts that does justice to our intuitions about their use, or which carves reality at the joints, is part of what it means to do philosophy.
Q: Isn't Philosophy, or the study of philosophy, supposed to tell you the Meaning of Life?
The concern expressed here IS one that is taken up by philosophers, some quite explicitly. The answers they give cover quite a range of possibilities, but some agreement among these disparate answers can be seen.
Viewed from far enough away (a distant planet, say), all life, or just human life, can seem meaningless, even absurd. Our lives are just a temporal blink in the cosmic time scale, effecting only an infinitesimally minute corner of a vast universe. And these are facts of which we are painfully aware.
Some have thought that this unease could be solved vicariously by positing a God, or Gods, whose own purposes could be attached to our lives. The point of existence would be then to adopt these purposes as our own. Others reject this solution, either because they find no reason to accept the existence of such deities, or because they insist that a truly meaningful life cannot be achieved vicariously.
Still others shrug off the sentiments which precipitate the problem. Bertrand Russell, for one, claims that no one really cares that the universe as we know it will end in 10 billion more years--and thus lacks an ultimate purpose. Nor should we care about the fact that our lives will come to a more or less abrupt end within the next century. One might wish to argue that my doing good acts can only make sense if they are done in order to balance some cosmic account of my character (and to gain some ultimate reward). But anyone who does make such an argument should also be able to find motivation to act in the acts themselves--after all, by hypothesis, they have already determined that they are the right thing to do.
In short, one might argue that the questions regarding life's purpose or meaning are badly framed. "What is its purpose?" or "Where is it to be found?" are all too easily answered: "there isn't one", and "nowhere", respectively. None of this implies, however, that an individual's life cannot be filled with purpose, meaning, and goals. It is only that these purposes are not something we discover in the world, as if they were new continents. Rather they are created and maintained by each individual. You make your life meaningful by the adoption of certain goals which you take to be important. You may even decide that the worship of God is what gives meaning to your life. But this works only when you recognize that you are making a decision, and not a discovery.