Stefanie Krug - Profile on Academia.edu (original) (raw)

Papers by Stefanie Krug

bioRxiv (Cold Spring Harbor Laboratory), Apr 8, 2024

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior ... more The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but 5 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these 4 DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC genes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

JCI insight, Oct 22, 2023

The Journal of Infectious Diseases, May 9, 2023

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in ... more Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA -/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA +/-) and homozygous (GBA +/+ ) isogenic lines. Macrophages derived from GBA -/-, GBA +/-and GBA +/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA -/-, GBA +/-and GBA +/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis. Keywords. CRISPR/Cas9 editing; Gaucher disease; human-induced pluripotent stem cells (hiPSCs); macrophage motility; Mycobacterium tuberculosis uptake and multiplication. Macrophage GCase enzymatic assays, phagocytosis activity, and motility were measured as described in the Supplementary Materials.

Nature Communications, Dec 8, 2023

The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens tre... more The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens treatment durations by several months despite being only weakly bactericidal. Intriguingly, PZA is also an anti-inflammatory molecule shown to specifically reduce inflammatory cytokine signaling and lesion activity in TB patients. However, the target and clinical importance of PZA's host-directed activity during TB therapy remain unclear. Here, we identify the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1), a pro-inflammatory master regulator strongly activated in TB, as a functionally relevant host target of PZA. We show that PZA inhibits PARP1 enzymatic activity in macrophages and in mice where it reverses TB-induced PARP1 activity in lungs to uninfected levels. Utilizing a PZA-resistant mutant, we demonstrate that PZA's immunemodulatory effects are PARP1-dependent but independent of its bactericidal activity. Importantly, PZA's bactericidal efficacy is impaired in PARP1-deficient mice, suggesting that immune modulation may be an integral component of PZA's antitubercular activity. In addition, adjunctive PARP1 inhibition dramatically reduces inflammation and lesion size in mice and may be a means to reduce lung damage and shorten TB treatment duration. Together, these findings provide insight into PZA's mechanism of action and the therapeutic potential of PARP1 inhibition in the treatment of TB. M. tuberculosis (M.tb) is a highly successful human pathogen able to establish lifelong infections in spite of powerful inflammatory immune responses 1,2 . M.tb is first encountered by airway-resident myeloid cells which recruit and activate immune cells by initiating transcription factor signaling, notably NF-κB, to produce critical chemo-and cytokines, including TNFα, . TNFα and IL-12 then stimulate the antimicrobial activity of macrophages, CD4 + Th1 cell differentiation, and production of the type 2 interferon IFNγ 4 . With the accumulation of M.tb antigen-specific CD4 + T cells, the host effectively restricts M.tb proliferation but is unable to clear the infection and resorts to surrounding infected foci with granulomatous structures to prevent further dissemination 4,5 . However, granulomas are associated with TB transmission, lung damage, poor drug penetration, and high antibiotic tolerance that make persisting bacilli difficult to kill .

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in ... more Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase)GBAgene, which result in macrophage dysfunction. To investigate whether correction ofGBAmutations restores normal function to Gaucher macrophages, we performed CRISPR editing of homozygous L444P (1448T→C)GBAmutation in Type 2 GD (GBA-/-) hiPSCs, which yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived fromGBA-/-,GBA+/- andGBA+/+ hiPSCs, were compared for GCase enzymatic activity, motility, and phagocytosis, all of which showed thatGBAmutation correction restores normal macrophage functions. Furthermore, we investigated whether lysosomal disorders drive susceptibility toMycobacterium tuberculosis, by infectingGBA-/-,GBA+/- andGBA+/+ macrophages with the virulent H37Rv lab strain. The results showed that impaired mobility and phagocytic activity of Gaucher macrophages, correlated with reduced levels of TB engulfmen...

Adjunctive Integrated Stress Response Inhibition Accelerates Tuberculosis Clearance in Mice

MBio, Feb 28, 2023

The Role of PARP1 and PARP Inhibition in Tuberculosis

Johns Hopkins University, Mar 18, 2021

Tuberculosis (TB) is a devastating infectious disease that is challenging to treat and responsibl... more Tuberculosis (TB) is a devastating infectious disease that is challenging to treat and responsible for over a million human deaths every year. TB elicits a multifaceted host response that can contain the infection at the cost of inflammation, tissue destruction and prolonged treatment durations. Consequently, one strategy to improve the duration and outcome of TB therapy is to combine antibacterial with host-directed agents. Interestingly, the critical TB drug pyrazinamide (PZA) has both antibiotic and immune-modulatory activities but its mechanism of action remains poorly understood. Insight into PZA’s sterilizing mechanism may lead to the development of desperately needed improved TB treatment options. In my dissertation work, I identified the eukaryotic master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as the first host target of PZA. PARP1 regulates fundamental cellular processes and immune functions but its role in the TB host response had not yet been characterized. I found PARP1 to be robustly activated in the lungs of TB-infected mice but potently inhibited by clinically relevant concentrations of PZA. Using structure- and activity-based assays, I demonstrated that PZA is indeed a novel PARP inhibitor that reversed TB-induced PAR formation in mice to uninfected levels. Remarkably, the therapeutic efficacy of PZA was reduced in PARP1-deficient mice, indicating that PARP inhibition may be central to PZA’s mechanism of action. In addition, adjunctive PARP inhibition dramatically reduced lung inflammation in infected mice and may be a promising new therapeutic approach that could mitigate TB-associated lung damage. Unexpectedly, I discovered sexually divergent effects of PARP1 deficiency in TB mouse models that were protective in females, resulting in accelerated bacterial clearance and prolonged survival, but detrimental in males, as indicated by impaired bacterial containment and exacerbated lung inflammation. PARP1 deficiency also eliminated sex differences in TB cytokine responses, suggesting that PARP1 may contribute to the TB male bias by specifically enhancing male proinflammatory responses. In conclusion, my dissertation work identified PARP1 as the first molecular driver of TB sex differences and a novel host target of PZA involved in its therapeutic efficacy. Adjunctive PARP inhibition is a promising new host-directed TB therapy that may improve patient outcome

Journal of Comparative Neurology, 2013

Naturally occurring cell death is essential to the development of the mammalian nervous system. A... more Naturally occurring cell death is essential to the development of the mammalian nervous system. Although the importance of developmental cell death has been appreciated for decades, there is no comprehensive account of cell death across brain areas in the mouse. Moreover, several regional sex differences in cell death have been described for the ventral forebrain and hypothalamus, but it is not known how widespread the phenomenon is. We used immunohistochemical detection of activated caspase-3 to identify dying cells in the brains of male and female mice from postnatal day (P) 1 to P11. Cell death density, total number of dying cells, and regional volume were determined in 16 regions of the hypothalamus and ventral forebrain (the anterior hypothalamus, arcuate nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and ventromedial nucleus of the hypothalamus; the basolateral, central, and medial amygdala; the lateral and principal nuclei of the bed nuclei of the stria terminalis; the caudate-putamen; the globus pallidus; the lateral septum; and the islands of Calleja). All regions showed a significant effect of age on cell death. The timing of peak cell death varied between P1 to P7, and the average rate of cell death varied tenfold among regions. Several significant sex differences in cell death and/or regional volume were detected. These data address large gaps in the developmental literature and suggest interesting region-specific differences in the prevalence and timing of cell death in the hypothalamus and ventral forebrain.

Lengthy tuberculosis (TB) treatment is required to address the ability of a subpopulation of pers... more Lengthy tuberculosis (TB) treatment is required to address the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine construct involving fusion of the relMtb gene with the immature dendritic cell-targeting gene encoding chemokine MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that the intramuscular MIP-3α/relMtb (fusion) vaccine potentiates isoniazid activity more than a similar DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 colony-forming units, P=0.0001), inducing pronounced Mtb-protective immune signatures. The intranasal fusion vaccine, an approach combining relMtb fusion to MIP-3α and intranasal delivery, demonstrated the greatest therapeutic ...

Three animals born to homozygous tau mutant (tss, “super short”) Syrian hamsters showed extremely... more Three animals born to homozygous tau mutant (tss, “super short”) Syrian hamsters showed extremely short free-running periods of locomotor activity (tDD of approximately 17.8 hours). Inbreeding produced 33 such “super duper ” animals, which had a tDD of 18.09 ± 0.05 hours, which was shorter than that of tss hamsters (20.66 ± 0.07 hours, p < 0.001). To test the hypothesis that a gene (Duper) is responsible for a 2-hour shortening of tDD, we backcrossed super duper hamsters to unrelated tss animals. The F1 pups uniformly had a tDD similar to that of tss hamsters (19.89 ± 0.15 hours), but F2 animals showed a 1:1 ratio of the 18- to 20-hour phenotypes. In contrast, the F1 of a cross between super duper hamsters and tss animals presumed heterozygous for duper showed a 1:1 ratio of 18- to 20-hour phenotypes, and inbreeding of the super duper F1 offspring uni-formly produced F2 pups with extremely short tDD (17.86 ± 0.5 hours). We isolated the duper mutation on a wild-type background thr...

Duper: a mutation that shortens hamster circadian period

Adjunctive inhibition of the integrated stress response pathway accelerates bacterial clearance in a mouse model of tuberculosis

ABSTRACTTuberculosis (TB) is a devastating infectious disease that continues to cause millions of... more ABSTRACTTuberculosis (TB) is a devastating infectious disease that continues to cause millions of human deaths every year. Even though most cases of TB can be cured with a 6-month antibiotic combination therapy, these long treatment durations have led to the emergence of multi-drug resistance and pose a major hurdle to global TB control. Despite numerous advances in TB drug development, a substantially shortened treatment time has yet to be achieved. Given the rise in antibiotic resistance, an alternative strategy to the direct targeting of M. tuberculosis (M.tb) is the development of host-directed therapies (HDTs) that promote bacterial clearance and/or lung health when given adjunctive to standard TB antibiotics. We recently discovered that a small molecule inhibitor of the Integrated Stress Response (ISR), which is abnormally activated in TB and associated with the formation of necrotic granulomas, reduced M.tb numbers and lung inflammation in mice. Here, we evaluated the therape...

Cell death atlas of the postnatal mouse brain: Effects of age and sex

Social Neuroscience, 2011

Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths ann... more Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths annually that has a poorly understood male bias. Elucidating the basis of this male bias may enable precision medicine interventions for TB treatment and prevention. Here, we identify the master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as a driver of TB sex differences. We found that infection with M. tuberculosis (M. tb) triggers robust PARP activation in mouse lungs, suggesting that PARP1 activation is a fundamental host response to TB. Remarkably, PARP1 deletion abolished known sex differences in TB cytokine responses and blunted the early induction of TNFα, IL-1ß, IFNγ, MCP-1, and IL-6, particularly in male mice. In contrast, PARP1 was required for IL-10 induction in male or female mice. PARP1 deletion was protective against TB in female mice, resulting in significantly prolonged survival and reduced bacterial burden, but impaired TB containment in male mice. Our findings indicat...

Frontiers in Immunology

Following infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), mo... more Following infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), most human hosts are able to contain the infection and avoid progression to active TB disease through expression of a balanced, homeostatic immune response. Proinflammatory mechanisms aiming to kill, slow and sequester the pathogen are key to a successful host response. However, an excessive or inappropriate pro-inflammatory response may lead to granuloma enlargement and tissue damage, which may prolong the TB treatment duration and permanently diminish the lung function of TB survivors. The host also expresses certain anti-inflammatory mediators which may play either beneficial or detrimental roles depending on the timing of their deployment. The balance between the timing and expression levels of pro- and anti-inflammatory responses plays an important role in the fate of infection. Interestingly, M. tuberculosis appears to manipulate both sides of the human immune response to remodel the...

Pharmacologic Exhaustion of Suppressor Cells with Tasquinimod Enhances Bacterial Clearance during TB

American Journal of Respiratory and Critical Care Medicine

respiratory support during neonatal intensive care with existing monitoring tools focusing on ven... more respiratory support during neonatal intensive care with existing monitoring tools focusing on ventilation alone. A further strength of this case report is the lack of any adjunctive respiratory support during the measurements. EIT demonstrated an almost complete lack of tidal ventilation in the left lung. EIT has been repeatedly shown to be able to differentiate single-lung ventilation in animal and human reports (5). Interestingly, EIT detected some residual tidal ventilation that clinicians would not have seen without the EIT recordings. The pulmonary artery infarct likely occurred after the pseudoglandular period and airway development. The residual ventilation likely represents bronchial tidal air movement and collateral ventilation via pores of Kohn. This suggests that EIT is able to image complex ventilation patterns within the lung at a resolution not currently possible within a bedside tool. EIT holds potential in monitoring ventilation and perfusion development in conditions characterized by unilateral lung differences and altered pulmonary blood flow, such as congenital diaphragmatic hernia and after cardiac surgery, over longer time periods. To date, the use of EIT to measure lung perfusion has been limited to human observations in which the perfusion state was not known (2) or animal models with selective occlusion of a single pulmonary artery and injection of an electrical impedance contrast agent (3). Our data support the preclinical data demonstrating the ability of EIT to selectively measure lung perfusion. The clear differentiation between the heartbeat-related signals within the left and right lung suggests that the cardiac-domain impedance measures within the anatomical regions of the chest containing lung are not exclusively due to heart movement being transmitted through lung regions. Conclusions. This case report describes the first report of EIT to measure perfusion and ventilation patterns in a newborn with a known loss of both in a single lung. EIT was able to demonstrate the perfusion and ventilation defects found on static radiological imaging. Importantly, EIT recordings within the heart-rate domain were independent of heart movement and at a respiratory resolution able to identify small areas of tidal ventilation. This suggests that EIT may hold promise as the first tool able to dynamically measure ventilation–perfusion matching over time in infants. n

Endocrinology, 2013

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and anteroventral perive... more The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and anteroventral periventricular nucleus of the hypothalamus (AVPV) are sexually dimorphic, hormone-sensitive forebrain regions. Here we report a profound sex difference in estrogen receptor-α (ERα) immunoreactivity (IR) in the BNSTp, with robust ERα IR in females and the near absence of labeling in males. This sex difference is due to the suppression of ERα IR by testicular hormones in adulthood: it was not present at birth and was not altered by neonatal treatment of females with estradiol; gonadectomy of adult males increased ERα IR to that of females, whereas gonadectomy of adult females had no effect. Treating gonadally intact males with an aromatase inhibitor partially feminized ERα IR in the BNSTp, suggesting that testicular suppression required aromatization. By contrast, in AVPV we found a modest sex difference in ERα IR that was relatively insensitive to steroid manipulations in adulthood. ERα IR in ...

Effects of the Duper Mutation on Circadian Responses to Light

Journal of Biological Rhythms, 2011

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)... more The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.

Duper: A Mutation that Shortens Hamster Circadian Period

Journal of Biological Rhythms, 2011

Three animals born to homozygous tau mutant (τ(ss), &amp;amp;amp;amp;amp;amp;amp;amp;quot;sup... more Three animals born to homozygous tau mutant (τ(ss), &amp;amp;amp;amp;amp;amp;amp;amp;quot;super short&amp;amp;amp;amp;amp;amp;amp;amp;quot;) Syrian hamsters showed extremely short free-running periods of locomotor activity (τ(DD) of approximately 17.8 hours). Inbreeding produced 33 such &amp;amp;amp;amp;amp;amp;amp;amp;quot;super duper&amp;amp;amp;amp;amp;amp;amp;amp;quot; animals, which had a τ(DD) of 18.09 ± 0.05 hours, which was shorter than that of τ(ss) hamsters (20.66 ± 0.07 hours, p &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). To test the hypothesis that a gene (Duper) is responsible for a 2-hour shortening of τ(DD), we backcrossed super duper hamsters to unrelated τ(ss) animals. The F(1) pups uniformly had a τ(DD) similar to that of τ(ss) hamsters (19.89 ± 0.15 hours), but F(2) animals showed a 1:1 ratio of the 18- to 20-hour phenotypes. In contrast, the F(1) of a cross between super duper hamsters and τ(ss) animals presumed heterozygous for duper showed a 1:1 ratio of 18- to 20-hour phenotypes, and inbreeding of the super duper F(1) offspring uniformly produced F(2) pups with extremely short τ(DD) (17.86 ± 0.5 hours). We isolated the duper mutation on a wild-type background through crossing of super duper with wild-type animals. Restriction digests identified short-period F(2) pups that lack the mutant CK1ε allele, and these animals had a mean τ(DD) of 23.11 ± 0.04 hours. τ(DD) of duper hamsters born and raised in DD was significantly shorter than in hamsters raised in 14L:10D (21.92 ± 0.12 hours, p &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001). τ(DD) shortened twice as much in τ(s) and τ(ss) hamsters than in wild-type animals that were homozygous for duper, indicating the presence of epistatic interactions. Assortment of phenotypes in the F(2) generation fit the expected distribution for expression of duper as recessive (χ(2) = 6.41, p &amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.1). Neither CK1ε nor CK1δ coding region base sequences differed between super duper and τ(ss) hamsters. The growth rate of super duper mutants is similar to that of τ(ss) animals but slightly but significantly reduced at particular postweaning time points. We conclude that duper represents a new mutation that substantially reduces τ(DD) and has significant effects on physiology and metabolism.

bioRxiv (Cold Spring Harbor Laboratory), Apr 8, 2024

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior ... more The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but 5 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these 4 DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC genes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

JCI insight, Oct 22, 2023

The Journal of Infectious Diseases, May 9, 2023

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in ... more Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA -/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA +/-) and homozygous (GBA +/+ ) isogenic lines. Macrophages derived from GBA -/-, GBA +/-and GBA +/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA -/-, GBA +/-and GBA +/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis. Keywords. CRISPR/Cas9 editing; Gaucher disease; human-induced pluripotent stem cells (hiPSCs); macrophage motility; Mycobacterium tuberculosis uptake and multiplication. Macrophage GCase enzymatic assays, phagocytosis activity, and motility were measured as described in the Supplementary Materials.

Nature Communications, Dec 8, 2023

The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens tre... more The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens treatment durations by several months despite being only weakly bactericidal. Intriguingly, PZA is also an anti-inflammatory molecule shown to specifically reduce inflammatory cytokine signaling and lesion activity in TB patients. However, the target and clinical importance of PZA's host-directed activity during TB therapy remain unclear. Here, we identify the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1), a pro-inflammatory master regulator strongly activated in TB, as a functionally relevant host target of PZA. We show that PZA inhibits PARP1 enzymatic activity in macrophages and in mice where it reverses TB-induced PARP1 activity in lungs to uninfected levels. Utilizing a PZA-resistant mutant, we demonstrate that PZA's immunemodulatory effects are PARP1-dependent but independent of its bactericidal activity. Importantly, PZA's bactericidal efficacy is impaired in PARP1-deficient mice, suggesting that immune modulation may be an integral component of PZA's antitubercular activity. In addition, adjunctive PARP1 inhibition dramatically reduces inflammation and lesion size in mice and may be a means to reduce lung damage and shorten TB treatment duration. Together, these findings provide insight into PZA's mechanism of action and the therapeutic potential of PARP1 inhibition in the treatment of TB. M. tuberculosis (M.tb) is a highly successful human pathogen able to establish lifelong infections in spite of powerful inflammatory immune responses 1,2 . M.tb is first encountered by airway-resident myeloid cells which recruit and activate immune cells by initiating transcription factor signaling, notably NF-κB, to produce critical chemo-and cytokines, including TNFα, . TNFα and IL-12 then stimulate the antimicrobial activity of macrophages, CD4 + Th1 cell differentiation, and production of the type 2 interferon IFNγ 4 . With the accumulation of M.tb antigen-specific CD4 + T cells, the host effectively restricts M.tb proliferation but is unable to clear the infection and resorts to surrounding infected foci with granulomatous structures to prevent further dissemination 4,5 . However, granulomas are associated with TB transmission, lung damage, poor drug penetration, and high antibiotic tolerance that make persisting bacilli difficult to kill .

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in ... more Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase)GBAgene, which result in macrophage dysfunction. To investigate whether correction ofGBAmutations restores normal function to Gaucher macrophages, we performed CRISPR editing of homozygous L444P (1448T→C)GBAmutation in Type 2 GD (GBA-/-) hiPSCs, which yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived fromGBA-/-,GBA+/- andGBA+/+ hiPSCs, were compared for GCase enzymatic activity, motility, and phagocytosis, all of which showed thatGBAmutation correction restores normal macrophage functions. Furthermore, we investigated whether lysosomal disorders drive susceptibility toMycobacterium tuberculosis, by infectingGBA-/-,GBA+/- andGBA+/+ macrophages with the virulent H37Rv lab strain. The results showed that impaired mobility and phagocytic activity of Gaucher macrophages, correlated with reduced levels of TB engulfmen...

Adjunctive Integrated Stress Response Inhibition Accelerates Tuberculosis Clearance in Mice

MBio, Feb 28, 2023

The Role of PARP1 and PARP Inhibition in Tuberculosis

Johns Hopkins University, Mar 18, 2021

Tuberculosis (TB) is a devastating infectious disease that is challenging to treat and responsibl... more Tuberculosis (TB) is a devastating infectious disease that is challenging to treat and responsible for over a million human deaths every year. TB elicits a multifaceted host response that can contain the infection at the cost of inflammation, tissue destruction and prolonged treatment durations. Consequently, one strategy to improve the duration and outcome of TB therapy is to combine antibacterial with host-directed agents. Interestingly, the critical TB drug pyrazinamide (PZA) has both antibiotic and immune-modulatory activities but its mechanism of action remains poorly understood. Insight into PZA’s sterilizing mechanism may lead to the development of desperately needed improved TB treatment options. In my dissertation work, I identified the eukaryotic master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as the first host target of PZA. PARP1 regulates fundamental cellular processes and immune functions but its role in the TB host response had not yet been characterized. I found PARP1 to be robustly activated in the lungs of TB-infected mice but potently inhibited by clinically relevant concentrations of PZA. Using structure- and activity-based assays, I demonstrated that PZA is indeed a novel PARP inhibitor that reversed TB-induced PAR formation in mice to uninfected levels. Remarkably, the therapeutic efficacy of PZA was reduced in PARP1-deficient mice, indicating that PARP inhibition may be central to PZA’s mechanism of action. In addition, adjunctive PARP inhibition dramatically reduced lung inflammation in infected mice and may be a promising new therapeutic approach that could mitigate TB-associated lung damage. Unexpectedly, I discovered sexually divergent effects of PARP1 deficiency in TB mouse models that were protective in females, resulting in accelerated bacterial clearance and prolonged survival, but detrimental in males, as indicated by impaired bacterial containment and exacerbated lung inflammation. PARP1 deficiency also eliminated sex differences in TB cytokine responses, suggesting that PARP1 may contribute to the TB male bias by specifically enhancing male proinflammatory responses. In conclusion, my dissertation work identified PARP1 as the first molecular driver of TB sex differences and a novel host target of PZA involved in its therapeutic efficacy. Adjunctive PARP inhibition is a promising new host-directed TB therapy that may improve patient outcome

Journal of Comparative Neurology, 2013

Naturally occurring cell death is essential to the development of the mammalian nervous system. A... more Naturally occurring cell death is essential to the development of the mammalian nervous system. Although the importance of developmental cell death has been appreciated for decades, there is no comprehensive account of cell death across brain areas in the mouse. Moreover, several regional sex differences in cell death have been described for the ventral forebrain and hypothalamus, but it is not known how widespread the phenomenon is. We used immunohistochemical detection of activated caspase-3 to identify dying cells in the brains of male and female mice from postnatal day (P) 1 to P11. Cell death density, total number of dying cells, and regional volume were determined in 16 regions of the hypothalamus and ventral forebrain (the anterior hypothalamus, arcuate nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and ventromedial nucleus of the hypothalamus; the basolateral, central, and medial amygdala; the lateral and principal nuclei of the bed nuclei of the stria terminalis; the caudate-putamen; the globus pallidus; the lateral septum; and the islands of Calleja). All regions showed a significant effect of age on cell death. The timing of peak cell death varied between P1 to P7, and the average rate of cell death varied tenfold among regions. Several significant sex differences in cell death and/or regional volume were detected. These data address large gaps in the developmental literature and suggest interesting region-specific differences in the prevalence and timing of cell death in the hypothalamus and ventral forebrain.

Lengthy tuberculosis (TB) treatment is required to address the ability of a subpopulation of pers... more Lengthy tuberculosis (TB) treatment is required to address the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine construct involving fusion of the relMtb gene with the immature dendritic cell-targeting gene encoding chemokine MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that the intramuscular MIP-3α/relMtb (fusion) vaccine potentiates isoniazid activity more than a similar DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 colony-forming units, P=0.0001), inducing pronounced Mtb-protective immune signatures. The intranasal fusion vaccine, an approach combining relMtb fusion to MIP-3α and intranasal delivery, demonstrated the greatest therapeutic ...

Three animals born to homozygous tau mutant (tss, “super short”) Syrian hamsters showed extremely... more Three animals born to homozygous tau mutant (tss, “super short”) Syrian hamsters showed extremely short free-running periods of locomotor activity (tDD of approximately 17.8 hours). Inbreeding produced 33 such “super duper ” animals, which had a tDD of 18.09 ± 0.05 hours, which was shorter than that of tss hamsters (20.66 ± 0.07 hours, p < 0.001). To test the hypothesis that a gene (Duper) is responsible for a 2-hour shortening of tDD, we backcrossed super duper hamsters to unrelated tss animals. The F1 pups uniformly had a tDD similar to that of tss hamsters (19.89 ± 0.15 hours), but F2 animals showed a 1:1 ratio of the 18- to 20-hour phenotypes. In contrast, the F1 of a cross between super duper hamsters and tss animals presumed heterozygous for duper showed a 1:1 ratio of 18- to 20-hour phenotypes, and inbreeding of the super duper F1 offspring uni-formly produced F2 pups with extremely short tDD (17.86 ± 0.5 hours). We isolated the duper mutation on a wild-type background thr...

Duper: a mutation that shortens hamster circadian period

Adjunctive inhibition of the integrated stress response pathway accelerates bacterial clearance in a mouse model of tuberculosis

ABSTRACTTuberculosis (TB) is a devastating infectious disease that continues to cause millions of... more ABSTRACTTuberculosis (TB) is a devastating infectious disease that continues to cause millions of human deaths every year. Even though most cases of TB can be cured with a 6-month antibiotic combination therapy, these long treatment durations have led to the emergence of multi-drug resistance and pose a major hurdle to global TB control. Despite numerous advances in TB drug development, a substantially shortened treatment time has yet to be achieved. Given the rise in antibiotic resistance, an alternative strategy to the direct targeting of M. tuberculosis (M.tb) is the development of host-directed therapies (HDTs) that promote bacterial clearance and/or lung health when given adjunctive to standard TB antibiotics. We recently discovered that a small molecule inhibitor of the Integrated Stress Response (ISR), which is abnormally activated in TB and associated with the formation of necrotic granulomas, reduced M.tb numbers and lung inflammation in mice. Here, we evaluated the therape...

Cell death atlas of the postnatal mouse brain: Effects of age and sex

Social Neuroscience, 2011

Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths ann... more Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths annually that has a poorly understood male bias. Elucidating the basis of this male bias may enable precision medicine interventions for TB treatment and prevention. Here, we identify the master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as a driver of TB sex differences. We found that infection with M. tuberculosis (M. tb) triggers robust PARP activation in mouse lungs, suggesting that PARP1 activation is a fundamental host response to TB. Remarkably, PARP1 deletion abolished known sex differences in TB cytokine responses and blunted the early induction of TNFα, IL-1ß, IFNγ, MCP-1, and IL-6, particularly in male mice. In contrast, PARP1 was required for IL-10 induction in male or female mice. PARP1 deletion was protective against TB in female mice, resulting in significantly prolonged survival and reduced bacterial burden, but impaired TB containment in male mice. Our findings indicat...

Frontiers in Immunology

Following infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), mo... more Following infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), most human hosts are able to contain the infection and avoid progression to active TB disease through expression of a balanced, homeostatic immune response. Proinflammatory mechanisms aiming to kill, slow and sequester the pathogen are key to a successful host response. However, an excessive or inappropriate pro-inflammatory response may lead to granuloma enlargement and tissue damage, which may prolong the TB treatment duration and permanently diminish the lung function of TB survivors. The host also expresses certain anti-inflammatory mediators which may play either beneficial or detrimental roles depending on the timing of their deployment. The balance between the timing and expression levels of pro- and anti-inflammatory responses plays an important role in the fate of infection. Interestingly, M. tuberculosis appears to manipulate both sides of the human immune response to remodel the...

Pharmacologic Exhaustion of Suppressor Cells with Tasquinimod Enhances Bacterial Clearance during TB

American Journal of Respiratory and Critical Care Medicine

respiratory support during neonatal intensive care with existing monitoring tools focusing on ven... more respiratory support during neonatal intensive care with existing monitoring tools focusing on ventilation alone. A further strength of this case report is the lack of any adjunctive respiratory support during the measurements. EIT demonstrated an almost complete lack of tidal ventilation in the left lung. EIT has been repeatedly shown to be able to differentiate single-lung ventilation in animal and human reports (5). Interestingly, EIT detected some residual tidal ventilation that clinicians would not have seen without the EIT recordings. The pulmonary artery infarct likely occurred after the pseudoglandular period and airway development. The residual ventilation likely represents bronchial tidal air movement and collateral ventilation via pores of Kohn. This suggests that EIT is able to image complex ventilation patterns within the lung at a resolution not currently possible within a bedside tool. EIT holds potential in monitoring ventilation and perfusion development in conditions characterized by unilateral lung differences and altered pulmonary blood flow, such as congenital diaphragmatic hernia and after cardiac surgery, over longer time periods. To date, the use of EIT to measure lung perfusion has been limited to human observations in which the perfusion state was not known (2) or animal models with selective occlusion of a single pulmonary artery and injection of an electrical impedance contrast agent (3). Our data support the preclinical data demonstrating the ability of EIT to selectively measure lung perfusion. The clear differentiation between the heartbeat-related signals within the left and right lung suggests that the cardiac-domain impedance measures within the anatomical regions of the chest containing lung are not exclusively due to heart movement being transmitted through lung regions. Conclusions. This case report describes the first report of EIT to measure perfusion and ventilation patterns in a newborn with a known loss of both in a single lung. EIT was able to demonstrate the perfusion and ventilation defects found on static radiological imaging. Importantly, EIT recordings within the heart-rate domain were independent of heart movement and at a respiratory resolution able to identify small areas of tidal ventilation. This suggests that EIT may hold promise as the first tool able to dynamically measure ventilation–perfusion matching over time in infants. n

Endocrinology, 2013

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and anteroventral perive... more The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and anteroventral periventricular nucleus of the hypothalamus (AVPV) are sexually dimorphic, hormone-sensitive forebrain regions. Here we report a profound sex difference in estrogen receptor-α (ERα) immunoreactivity (IR) in the BNSTp, with robust ERα IR in females and the near absence of labeling in males. This sex difference is due to the suppression of ERα IR by testicular hormones in adulthood: it was not present at birth and was not altered by neonatal treatment of females with estradiol; gonadectomy of adult males increased ERα IR to that of females, whereas gonadectomy of adult females had no effect. Treating gonadally intact males with an aromatase inhibitor partially feminized ERα IR in the BNSTp, suggesting that testicular suppression required aromatization. By contrast, in AVPV we found a modest sex difference in ERα IR that was relatively insensitive to steroid manipulations in adulthood. ERα IR in ...

Effects of the Duper Mutation on Circadian Responses to Light

Journal of Biological Rhythms, 2011

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)... more The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.

Duper: A Mutation that Shortens Hamster Circadian Period

Journal of Biological Rhythms, 2011

Three animals born to homozygous tau mutant (τ(ss), &amp;amp;amp;amp;amp;amp;amp;amp;quot;sup... more Three animals born to homozygous tau mutant (τ(ss), &amp;amp;amp;amp;amp;amp;amp;amp;quot;super short&amp;amp;amp;amp;amp;amp;amp;amp;quot;) Syrian hamsters showed extremely short free-running periods of locomotor activity (τ(DD) of approximately 17.8 hours). Inbreeding produced 33 such &amp;amp;amp;amp;amp;amp;amp;amp;quot;super duper&amp;amp;amp;amp;amp;amp;amp;amp;quot; animals, which had a τ(DD) of 18.09 ± 0.05 hours, which was shorter than that of τ(ss) hamsters (20.66 ± 0.07 hours, p &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). To test the hypothesis that a gene (Duper) is responsible for a 2-hour shortening of τ(DD), we backcrossed super duper hamsters to unrelated τ(ss) animals. The F(1) pups uniformly had a τ(DD) similar to that of τ(ss) hamsters (19.89 ± 0.15 hours), but F(2) animals showed a 1:1 ratio of the 18- to 20-hour phenotypes. In contrast, the F(1) of a cross between super duper hamsters and τ(ss) animals presumed heterozygous for duper showed a 1:1 ratio of 18- to 20-hour phenotypes, and inbreeding of the super duper F(1) offspring uniformly produced F(2) pups with extremely short τ(DD) (17.86 ± 0.5 hours). We isolated the duper mutation on a wild-type background through crossing of super duper with wild-type animals. Restriction digests identified short-period F(2) pups that lack the mutant CK1ε allele, and these animals had a mean τ(DD) of 23.11 ± 0.04 hours. τ(DD) of duper hamsters born and raised in DD was significantly shorter than in hamsters raised in 14L:10D (21.92 ± 0.12 hours, p &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001). τ(DD) shortened twice as much in τ(s) and τ(ss) hamsters than in wild-type animals that were homozygous for duper, indicating the presence of epistatic interactions. Assortment of phenotypes in the F(2) generation fit the expected distribution for expression of duper as recessive (χ(2) = 6.41, p &amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.1). Neither CK1ε nor CK1δ coding region base sequences differed between super duper and τ(ss) hamsters. The growth rate of super duper mutants is similar to that of τ(ss) animals but slightly but significantly reduced at particular postweaning time points. We conclude that duper represents a new mutation that substantially reduces τ(DD) and has significant effects on physiology and metabolism.