Snake bite remains a common injury in endemic areas of Pakistan. A facility based observational study was undertaken to ascertain the incidence of snake bite, its mortality and the management in public health facilities located in the Thar Desert of Province of Sindh, Pakistan. A total of 771 cases of snake bite were reported to seven randomly selected health facilities during a one-year period. Most (75%) occurred during nighttime in summer, affecting primarily males (70%) who were bitten mostly on the lower extremities (83%). Five hundred and thirteen bites (66%) were identified as poisonous and 4 deaths were reported. Seventy two percent of patients reached the health facility within 24 hours and twenty percent within 6 hours and of being bitten. Snake bite remains an important problem in endemic areas of Pakistan. People do seek assistance at public health facilities, where appropriate treatment including anti snake venom is given.
Abstract Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αβ T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of MR1 restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in healthy individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with atypical MAIT cells and tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in healthy people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.
Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αβ T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis ( Mtb ) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.
Whole-genome sequence (WGS) analyses were employed to investigate the genomic epidemiology of extensively drug-resistant Klebsiella pneumoniae strains, focusing on the carbapenem resistance-encoding determinants, mobile genetic support, clonal and epidemiological relationships. A total of ten isolates were obtained from patients admitted to the intensive care unit (ICU) in a public hospital in South Africa. Five isolates were from rectal swabs of colonized patients and five from blood cultures of patients with invasive carbapenem-resistant infections. Following microbial identification and antibiotic susceptibility tests, the isolates were subjected to WGS on the Illumina MiSeq platform. All the isolates showed genotypic resistance to tested β-lactams (NDM-1, OXA-1, CTX-M-15, TEM-1B, SHV-1) and other antibiotics. All but one isolate belonged to the ST152 with a novel sequence type, ST3136, differing by a single-locus variant. The isolates had the same plasmid multilocus sequence type (IncF[K12:A-:B36]) and capsular serotype (KL149), supporting the epidemiological linkage between the clones. Resistance to carbapenems in the 10 isolates was conferred by the blaNDM-1 mediated by the acquisition of multi-replicon [ColRNAI, IncFIB(pB171), Col440I, IncFII, IncFIB(K) and IncFII(Yp)] p18-43_01 plasmid. These findings suggest that the acquisition of blaNDM-1-bearing plasmid structure (p18-43_01), horizontal transfer and clonal dissemination facilitate the spread of carbapenemases in South Africa. This emphasizes the importance of targeted infection control measures to prevent dissemination.
The mechanisms by which HIV increases susceptibility to tuberculosis and other respiratory infections are incompletely understood. We used transcriptomics of paired whole bronchoalveolar lavage cells (BLCs) and peripheral blood mononuclear cells to compare the effect of HIV at the lung mucosal surface and in peripheral blood. The majority of HIV-induced differentially expressed genes (DEGs) were specific to either the peripheral or lung mucosa compartments (1,307/1,404, 93%). Type I interferon signaling was the dominant signature of DEGs in HIV-positive blood but not in HIV-positive BLCs. DEGs in the HIV-positive BLCs were significantly enriched for infiltration with cytotoxic CD8+ T cells. Higher expression of type 1 interferon transcripts in peripheral CD8+ T cells and representative transcripts and proteins in BLCs-derived CD8+ T cells during HIV infection, including IFNG (IFN-gamma), GZMB (Granzyme B) and PDCD1 (PD-1), was confirmed by cell-subset specific transcriptional analysis and flow cytometry. Thus, we report that a whole transcriptomic approach revealed qualitatively distinct effects of HIV in blood and bronchoalveolar compartments. Further work exploring the impact of distinct type I interferon programs and functional features of CD8+ T cells infiltrating the lung mucosa during HIV infection may provide novel insights into HIV-induced susceptibility to respiratory pathogens.
A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.
Abstract Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2 + semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboflavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and mycobacterial infections in murine models. Here, we report that a population of pro-inflammatory TRAV1-2 + CD8 + T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary tuberculosis (TB). High-throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor-unique TRAV1-2 + MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These findings demonstrate enrichment of TRAV1-2 + CD8 + T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to Mycobacterium tuberculosis .
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. https://doi.org/10.7554/eLife.22028.001 eLife digest Every year, around two million people worldwide die from tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). The bacteria generally infect the lungs. In response, the immune system forms structures called granulomas that attempt to control and isolate the infecting pathogens. Granulomas consist of immune cells known as macrophages, which engulf the M. tuberculosis bacteria and isolate them in a cellular compartment where the bacteria either cannot grow or are killed. However, if a large number of macrophages in a granuloma die, the granuloma's core liquefies and the structure is coughed up into the airways, from where M. tuberculosis bacteria are transmitted to other people. But how do the bacteria manage to cause the extensive death of the cells that are supposed to control the infection? By imaging M. tuberculosis in human macrophages using time-lapse microscopy, Mahamed et al. reveal that the bacteria break down macrophage control by serially killing macrophages. M. tuberculosis cells first clump together and 'gang up' on a macrophage, which engulfs the clump and dies because the bacteria overwhelm it. This does not kill the bacteria, and they rapidly grow inside the dead macrophage. The dead cell is then cleaned up by another macrophage. However, the increasing number of bacteria inside the dead macrophage means that the new macrophage is even more likely to die than the first one. Hence, the bacteria use dead macrophages as fuel to grow on and as bait to attract the next immune cell. Overall, Mahamed et al. show that once a clump of M. tuberculosis initiates death of a single macrophage, it may lead to serial killing of other macrophages and a loss of control over the infection. An important next step will be to understand how the initial clump of bacteria is allowed to form. https://doi.org/10.7554/eLife.22028.002 Introduction Tuberculosis is characterized by the formation of granulomas, cellular structures which attempt to 'wall off' infection by surrounding it with cells of the immune system (Ramakrishnan, 2012; Russell, 2007; Russell et al., 2010). Granulomas, which have a defined anatomical structure as well as segregated expression of immune system related proteins within the structure (Marakalala et al., 2016), differentiate and mature independent of each other in infected tissues, most often the lung. The process of pulmonary granuloma formation is driven by macrophage phagocytosis of inhaled, viable Mtb, followed by extravasation of monocytes and T cells from the circulation and their accumulation at the site of infection (Ramakrishnan, 2012; Russell, 2007; Russell et al., 2010; Barry et al., 2009). However, granulomas do not always succeed in containing Mtb infection, and different granulomas in the same lung can control the infection or support the growth of the bacilli (Barry et al., 2009; Lenaerts et al., 2015; Lin et al., 2014; Kaplan et al., 2003). In the latter case, central necrosis within the granuloma, cavity formation, and breach into the airways, results in release of Mtb into the environment and transmission of the infection to another human host (Ramakrishnan, 2012; Russell, 2007; Russell et al., 2010; Barry et al., 2009). Thus, while Mtb is considered an intracellular pathogen infecting live cells, its proliferation during active pulmonary tuberculosis occurs in an environment containing many dead cells and cell remnants (Kaplan et al., 2003; Hunter, 2011; Hunter et al., 2007; Welsh et al., 2011; Irwin et al., 2015). Part of the host response to Mtb infection is the death of the infected phagocyte. Mtb-induced macrophage death has been observed to occur by apoptotic and non-apoptotic mechanisms. Apoptosis was reported to be protective against Mtb (Fratazzi et al., 1999; Gan et al., 2008; Keane et al., 2000; Molloy et al., 1994; Oddo et al., 1998; Behar et al., 2010). Evidence from mouse macrophages shows that protection is mediated through efferocytosis, the internalization of apoptotic cell fragments by macrophages, leading to the elimination of the Mtb inside the cellular fragments (Martin et al., 2012). Mtb was also observed to induce non-apoptotic cell death, differentiated from apoptosis by the lack of caspase activation (Lee et al., 2006), lack of DNA fragmentation (Keane et al., 2000; Park et al., 2006), and loss of membrane integrity (Park et al., 2006; Dobos et al., 2000). Mtb was not killed when necrotic cell death was induced by H2O2 (Molloy et al., 1994). Mtb is reported to initiate non-apoptotic cell death by the breakdown of mitochondrial membrane integrity (Duan et al., 2002), interference with host plasma membrane repair through inhibition of prostaglandin E2 (Divangahi et al., 2009; Divangahi et al., 2010), and secretion of a necrosis inducing toxin which kills macrophages by hydrolyzing NAD (Sun et al., 2015). The inflammatory effect of non-apoptotic cell death resulting from the immediate loss of membrane integrity upon death (Fink and Cookson, 2005) may allow for the recruitment of additional host phagocytes to the site of infection which may lead to infection spread (Clay et al., 2007). In contrast, control of Mtb through apoptosis and autophagy (Castillo et al., 2012; Gutierrez et al., 2004; Watson et al., 2012) may dampen the inflammatory response. Mtb infection is known to be controlled by the adaptive immune response (Cooper and Flynn, 1995; Flynn et al., 1995, 1992) which becomes effective several weeks after exposure (Wolf et al., 2008), though a study using intravital imaging observed limited T cell effector function in Mtb granulomas (Egen et al., 2011). The adaptive immune response controls Mtb by activating macrophages with factors such as interferon gamma (IFNγ) to kill or control the growth of the bacilli, and by targeting infected macrophages with cytotoxic lymphocytes (Cooper and Flynn, 1995; Flynn et al., 1995, 1992). However, given that different infection outcomes can coexist in the same lung, the source for variability (Russell et al., 2009) in infection outcomes at different foci is not well understood. Substantial variability in other biological systems results from positive feedback (Kaern et al., 2005; Sigal et al., 2006). Positive feedback involves a cascade of events such that once an event occurs, the probability that it will occur again is increased. To understand how an environment conducive to bacterial growth and host cell death is sustained, we investigated virulent Mtb infection dynamics in primary human macrophages in vitro using time-lapse microscopy over approximately 80 hr at 10 min resolution. We observed that macrophage internalization of Mtb aggregates led to the killing of the host cell, with the frequency of host cell death increasing with aggregate size. Uptake of single large aggregates showed a higher frequency of cell death relative to the uptake of a similar number of bacteria as multiple small aggregates, and the time to death was inversely correlated to aggregate size. Once the host cell was killed, Mtb was able to rapidly grow inside the dead infected macrophage regardless of whether the cells were monocyte derived macrophages (MDM) or alveolar macrophages, or whether the cells were exposed to IFNγ prior to infection. Growth inside dead cells was significantly faster than in the extracellular environment. Once host cell death occurred, other macrophages internalized the dead infected cells, and this rapidly led to their own death, forming a cell death cascade. These observations of a cycle of host cell death followed by bacterial replication could drive Mtb infection dynamics of necrosis and bacterial proliferation in lung granulomas of patients with active tuberculosis disease. Results The number and aggregation state of Mtb internalized by individual cells determines macrophage fate We infected human MDM with the virulent M. tuberculosis H37Rv strain labelled with red fluorescent protein (RFP) or mCherry expressed under the control of a constitutively active promoter (groEL and smyc' respectively). We used live cell imaging in Biosafety Level 3 containment over a period of approximately 80 hr to follow the outcome of infection in individual macrophages. MDM cell borders were determined by custom written image analysis software (Video 1, Materials and methods, Source code 1). Time of cell death was determined to be the time point at which movement of internal cellular structures ceased or the cell detached (Materials and methods). This method of death determination was validated in macrophages with the DNA intercalating dye DRAQ7, which only enters the cell when membrane integrity has been compromised due to cell death (Figure 1—figure supplement 1). To examine whether Mtb fluorescence is a valid measure of Mtb number, we tracked the increase in Mtb by fluorescence versus colony forming units (CFU) over 3 days of growth. We found a tight correspondence between the two measures (Figure 1—figure supplement 2), with an incremental increase in fluorescence translating to an incremental increase in the number of bacilli as measured by CFU. Hence, fluorescence measurements reflect Mtb numbers and an increase in Mtb fluorescence reflects Mtb growth. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Macrophage internalization of Mtb aggregates. Mtb infections of human macrophages were imaged by time-lapse microscopy at a resolution of 10 min between image acquisitions. Macrophage borders and locations were tracked using a custom-written, Matlab based image analysis code (Source code 1). The left panel of the montage is a movie of macrophage infection. The border of the analyzed macrophage is shown as a green outline. RFP-expressing Mtb are shown in red. Mtb internalized by the cell is marked by blue ellipses. Time is hours:minutes. Scale bar is 20 µm. The graph on right shows the fluorescence signal converted to bacterial numbers in the analyzed macrophage. Timing of internalizations of Mtb clumps containing varying numbers of bacilli as detected from the movie are shown as vertical red lines on the graph. Macrophage death is marked by a vertical black line of x's appearing at the point of cell death. https://doi.org/10.7554/eLife.22028.003 We asked whether the number of Mtb internalized by MDM, as observed in our time-lapse movies, affected the probability of macrophage death. The aggregation state of Mtb has been hypothesized to impact host-pathogen interactions (Orme, 2014; Sani et al., 2010). Mtb naturally aggregate, unless cultured with a nonionic detergent such as Tween 80 (Dubos and Middlebrook, 1948). To minimize modification of the outer surface of Mtb and its aggregation state (Sani et al., 2010), we grew Mtb without detergent for several replication cycles and gently broke up aggregates resulting from such growth into a heterogeneous population of bacterial aggregation states. Hence, MDM in a single experiment were infected by individual bacteria or multibacterial clumps of various sizes (Video 1). This enabled us to compare the consequences of MDM phagocytosis of different Mtb numbers and aggregation states in the same experiment. We converted Mtb fluorescent signal inside MDM to bacterial numbers by dividing the signal by the mean fluorescence of a single bacterium, with single Mtb obtained by filtering. We then ranked MDM from lowest to highest by the sum of Mtb internalized. This ranking gave the best separation in the frequency of cell death between the top 50% and bottom 50% of ranks (Figure 1—figure supplement 3). The raw data describing the outcome of the macrophage-Mtb interactions is presented in Figure 1—figure supplement 4. The full dataset contains the dynamics of macrophage Mtb internalization and fate of 759 MDM, with 720 infected cells and 39 uninfected bystander cells. (Figure 1—figure supplement 4B). To compare the death frequencies between cells internalizing different numbers of Mtb over the course of the imaging, we divided the dataset into ten groups of infected cells, where cells internalizing a similar number of Mtb were grouped together and where each group constituted 10% of the total number of infected cells. We also compared death frequency to a group of bystander cells which did not phagocytose any Mtb. We then determined the frequency of cell death in each group, and as well as the mean number of Mtb in the group. Macrophage fate was found to depend on the number of Mtb internalized. As the number of bacteria internalized increased, there was more cell death and it occurred sooner (Figure 1). Phagocytosis of small numbers of Mtb, less than approximately 10 bacilli, did not induce significantly more macrophage death than observed in uninfected bystander cells (Figure 1—figure supplement 5). Above 10 Mtb internalized but below approximately 30, there was a trend toward increased macrophage death relative to bystander cells, but the differences in frequencies were not significant (see Figure 1—figure supplement 5B for p-values and significance thresholds). The probability of death increased significantly relative to bystanders and cells with less than 10 Mtb when more than 30 Mtb were internalized per cell over the course of the movie, and a threshold for Mtb to induce death was clearly visible in Figure 1 at about 50 Mtb internalized, with differences relative to bystander and lightly infected cells becoming highly significant (Figure 1—figure supplement 5B). Figure 1 with 5 supplements see all Download asset Open asset Number of Mtb internalized determines probability of macrophage death. The death frequency for 720 infected MDM was graphed over time as a function of the sum of Mtb internalized (raw data provided in Figure 1—source data 1). To obtain death frequencies, MDM were divided into 10 groups according to the number of Mtb internalized. The frequency of dead cells was determined at 10 movie frame (1.7 hr) intervals for each group (x-axis), and plotted against the mean sum of Mtb internalized for the group (y-axis). Interpolation between points was performed to obtain a surface. The color scale represents the frequency of cell death from low (blue) to high (red). https://doi.org/10.7554/eLife.22028.004 Figure 1—source data 1 Intracellular Mtb fluorescence through time, Movie frames of Mtb phagocytosis, and MDM frame of death, if it occurs, for IFNγ untreated MDM. Used in Figures 1–6. https://doi.org/10.7554/eLife.22028.005 Download elife-22028-fig1-data1-v3.xlsx We examined whether the frequency of cell death was different if a cell internalized a given amount of Mtb in one pickup as opposed to over multiple pickups. In order to control for bacterial number, we limited the comparison to cells which internalized an average of approximately 50 Mtb (49.6 ± 18.9 Mtb for macrophages which phagocytosed multiple smaller aggregates, versus 49.4 ± 14.3 for macrophages which phagocytosed one large aggregate), an infection level where approximately one half of infected cells died by the end of our imaging period of 83 hr (Figure 1—figure supplement 5A). This infection level was chosen so as to increase sensitivity by excluding very highly infected cells, which died with a high frequency, as well as lightly infected cells, which did not die at an appreciably higher frequency relative to bystanders. We obtained a lower frequency of cell death when infection occurred by multiple smaller pickups compared to single large pickups (Figure 2). While 47% of the cells died when they accumulated Mtb in three or more internalizations, 71% of the cells died when they internalized a similar mean number of Mtb as one pickup. The difference was significant (p=5×10−4 by bootstrap). In addition to the summary of the results (Figure 2A), we also represent the individual cell histories (Figure 2B for multiple pickups and Figure 2C for single pickups). The data are inherently complex as individual macrophages are tracked, and these phagocytose different numbers of Mtb in different states at different times, and may or may not die as a result. We represent each cell as a line, with pickup events (denoted as circles or stars, where are circles are internalizations of cell-free Mtb, and stars are internalizations of dead infected cells, both color coded according to the number of Mtb internalized) at a location on the line corresponding to the time of pickup. If macrophage death occurs, line color changes from green to dark blue at the time of death. Figure 2 Download asset Open asset Internalization of single large aggregates is more cytotoxic than several smaller aggregates. (A) Fraction of dead MDM after phagocytosing one large aggregate of Mtb (black line) or multiple small aggregates (red line) with a similar cumulative sum of Mtb to the single internalizations, but with all aggregates being smaller than 50 bacilli each. The frequency of cell death was 47% for multiple internalizations (n = 47) and 71% for single internalizations (n = 62, p=5×10−4 by bootstrap). Individual cell fates are shown in (B) for cells internalizing multiple small aggregates, and (C) for cells internalizing a similar number of Mtb as single aggregates. The blue circles are internalizations of aggregates of 10 or fewer Mtb, red circles are clumps of 11–49 Mtb, and black circles are 50–81 Mtb. Line color changes from green to dark blue at time of death. Stars indicate internalizations of dead infected cells. An equal number of cells is shown in (B) and (C) to facilitate comparison. Mean number of Mtb phagocytosed was 49.6 ± 18.9 for MDM internalizing multiple small Mtb clumps, and 49.4 ± 14.3 for MDM internalizing a single large clump. https://doi.org/10.7554/eLife.22028.011 We asked whether time to cell death was dependent on pickup size. We therefore compared macrophages which internalized single clumps of Mtb during the first half of the imaging period, to increase the measurable time to death, if it occurred. In lightly infected cells (≤10 Mtb internalized as one aggregate) cell death was intermittent and not clearly linked to the internalization event. In contrast, the time to cell death appeared to be clearly linked to the time of pickup when the number of Mtb in the clump was large (Figure 3A). The time to cell death after internalization inversely correlated with the number of bacteria internalized in a single pickup (Figure 3B, R2 = 0.45, p=2×10−6), indicating that uptake of larger clumps led to faster onset of macrophage death. Figure 3 Download asset Open asset Mtb aggregate size determines timing of macrophage death. (A) MDMs which internalized exactly one clump before the halfway point of the movie were ranked based on the amount of Mtb internalized (n = 72 cells). The blue circles are internalizations of aggregates of 10 or fewer Mtb, red circles are clumps of 11–49 Mtb, and black circles are >50 Mtb. Line color changes from green to dark blue at time of death. Stars indicate internalizations of dead infected cells. (B) Time differences between internalization and death for cells that died as a function of log transformed bacterial number. R2 = 0.56, p=4×10−8(n = 39). https://doi.org/10.7554/eLife.22028.012 It has been previously reported that high multiplicity infection with Mtb leads to non-apoptotic cell death (Molloy et al., 1994; Lee et al., 2006; Park et al., 2006; Dobos et al., 2000; Repasy et al., 2013). To examine the mode of Mtb aggregate-induced cell death, we compared Mtb-induced cell death to that triggered by cisplatin, a chemotherapeutic agent that induces macrophage apoptosis by DNA damage (von Knethen et al., 1998), and the toxin nigericin, reported to induce macrophage cell death by pyroptosis following LPS priming (Warny and Kelly, 1999). In apoptosis, loss of membrane integrity is delayed to provide an opportunity for the removal of the dead cell (Fadok et al., 1992; Vermes et al., 1995). In pyroptosis, necrosis and other inflammatory cell death types, cell death involves the immediate loss of membrane integrity and spillage of cell contents (Fink and Cookson, 2005). We used DRAQ7 to track loss of membrane integrity, and cessation of internal movement to define the point of cell death (Figure 4A–B, Materials and methods). Mtb typically induced phagocyte cell death with immediate loss of membrane integrity (Video 2, Figure 4), similar to nigericin induced cell death (Video 3, Figure 4), and consistent with a previous study using A549 epithelial cells (Dobos et al., 2000). In contrast, cisplatin induced cell death was apparent significantly before incorporation of DRAQ7 (Video 4, Figure 4). Figure 4 Download asset Open asset Mtb induced macrophage death does not resemble apoptosis. (A) Cell death was caused by internalization of Mtb clumps (top row, Mtb in red), the pyroptosis inducers Nigericin+LPS (middle row), or the apoptosis inducer cisplatin (bottom row). Time of cell death was determined by the cessation of internal movement, and loss of membrane integrity by entry of the fluorescent dye DRAQ7 (green). (B) Quantitation of time of cell death and loss of membrane integrity. Black line is the Pearson correlation of within cell image pixels from one time-lapse frame to the next. Cell death as determined by cessation of internal cell movement is indicated by greater than threshold jump in pixel correlation (red vertical line). Loss of membrane integrity as detected by DRAQ7 is shown by the green line, (normalized to maximum signal) with blue vertical line representing time DRAQ7 levels cross an experimentally determined threshold. In the case of Mtb-induced cell death, the blue and red lines are superimposed. Each graph represents the cell shown in (A). (C) Median time difference (red line, with an interquartile range shown as blue box) between cell death and loss of membrane integrity for cells killed by Mtb (n = 119), 20 µM nigericin and 1 µg/ml LPS (n = 29) or 50 µM cisplatin (n = 76). ****p<0.001 by Kruskal-Wallis rank sum test corrected for multiple hypotheses. https://doi.org/10.7554/eLife.22028.013 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Death of an Mtb-infected macrophage. MDMs were infected with Mtb H37Rv-RFP (red) and imaged in the presence of the viability dye DRAQ7 (green). The entry of DRAQ7 corresponds to the time of cell death. Scale bar is 20 µm. https://doi.org/10.7554/eLife.22028.014 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Pyroptotic death of macrophages. Macrophages were sensitized with 1 µg/ml LPS for 3 hr, then treated with 20 µM nigericin to induce pyroptosis. Cells were imaged in the presence of DRAQ7 (green). https://doi.org/10.7554/eLife.22028.015 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Cisplatin-Induced cell death. Macrophages were treated with 50 µM cisplatin and imaged in the presence of DRAQ7 (green). Scale bar is 20 µm. https://doi.org/10.7554/eLife.22028.016 Mtb clumps are not eliminated by macrophage death and rapidly grow inside the dead cell We tracked the fate of Mtb in the dead cells. Mtb-induced macrophage death did not lead to the killing of the bacilli, and Mtb fluorescence levels increased in the dead cells (Figure 5A, Video 5). While RFP or mCherry fluorescence possibly persists after Mtb death, the increase in fluorescent signal is a clear indication of Mtb replication (Figure 1—figure supplement 2). Figure 5A is typical of the type of bacterial growth we observed: Mtb did not burst out of the cell to spread into the neighboring medium. Instead, the bacilli remained tightly associated with the dead host cell as a growing clump. Out of the 92 dead infected cells we evaluated, 90 showed positive growth rates while two had declining Mtb numbers (Figure 5B). Intracellular Mtb had a median doubling time, excluding the two negative values, of 24.7 hr (interquartile range 19 to 34 hr, Figure 5B inset). The extracellular growth rate of Mtb in the same experiments had a median doubling time of 36.1 hr (interquartile range 31 to 44 hr, Figure 5B inset), significantly slower than in dead cells (p=7×10−8, Wilcoxon rank sum test). We confirmed these results by CFU, quantifying the number of Mtb after three days of growth in dead MDM versus extracellular growth in MDM medium. We initiated growth with the same concentrated Mtb culture, which killed approximately 99% of the MDMs after one day, thus allowing Mtb to replicate in the dead macrophages for at least two days (Figure 5—figure supplement 1A). We recovered approximately an order of magnitude more Mtb by CFU after growth in dead cells versus after growth in the MDM extracellular medium (Figure 5—figure supplement 1B). Figure 5 with 1 supplement see all Download asset Open asset Mtb grows robustly inside dead macrophages. (A) In silico synchronized images of representative macrophages infected with Mtb and imaged before and after death. Each horizontal set of images represents the same macrophage over time, with the cell death event at the center image. On the left of the cell death event are images of the cell at regular intervals before cell death as a percentage of time the cell was imaged alive. On the right are images at regular intervals after death a percentage of time the cell was imaged dead. (B) Traces of Mtb growth in dead cells. Each trace represents the number of Mtb within the same dead MDM and is normalized to its maximum Mtb number and maximum length of time the dead cell was imaged before detachment or movie end. Minimum time for imaging dead cells was 10 hr. In total, 92 dead macrophages were analyzed, with 90 showing positive exponential slopes. The median doubling time for cells with positive slopes was 24.7 hr over five independent experiments. Inset: Doubling times of Mtb in dead cells excluding the two negative values (red, n = 90) and in the extracellular medium (black, n = 60). The median extracellular doubling time was 36.1 hr. https://doi.org/10.7554/eLife.22028.017 Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Mtb grows inside a dead macrophage treated with IFNγ prior to infection. The left panel of the montage is a movie of macrophage infection at a resolution of 10 min between image acquisitions. The border of the analyzed macrophage is shown as a green outline, and the presence of DRAQ7 is shown in blue. RFP-expressing Mtb are in red. Time is hours:minutes. Scale bar is 20 µm. The graph on right shows the fluorescence signal converted to bacterial numbers in the analyzed macrophage. Timing of macrophage death was determined by the lack of internal movement and confirmed by a DRAQ7 entry (vertical line of x's). https://doi.org/10.7554/eLife.22028.019 We next compared the growth of Mtb in dead cells to that in live infected cells. Live macrophages internalized Mtb throughout the imaging period, and hence an increase in Mtb fluorescence could be the result of either new Mtb internalized, or existing Mtb growing in the live cell. Therefore, we marked the time each macrophage internalized Mtb, and used a 10 hr tracking wind
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