Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP.

IMPORTANCE: In this paper, we describe novel inhibitors of cyclic dinucleotide phosphodiesterase enzymes from Mycobacterium tuberculosis (M.tb) (CdnP) and mammals (ENPP1). The phosphodiesterase enzymes hydrolyze cyclic dinucleotides, such as 2',3'-cyclic GMP-AMP and c-di-AMP, which are stimulator of interferon gene (STING) agonists. By blocking the hydrolysis of STING agonists, the cyclic GMP-AMP synthase (cGAS)-STING-IRF3 pathway is potentiated. There is strong evidence in tuberculosis and in cancer biology that potentiation of the cGAS-STING-IRF3 pathway leads to improved M.tb clearance and also improved antitumor responses in cancer. In addition to the identification of novel inhibitors and their biochemical characterization, we provide proof-of-concept evidence that our E-3 inhibitor potentiates the cGAS-STING-IRF3 pathway in both macrophage cell lines and also in primary human monocyte-derived macrophages.

Inhibition of host PARP1 contributes to the anti-inflammatory and antitubercular activity of pyrazinamide.

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 immune-modulatory 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.

MDA5 RNA-sensing pathway activation by Mycobacterium tuberculosis promotes innate immune subversion and pathogen survival.

Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis pathogenesis, we evaluated M. tuberculosis intracellular growth and innate immune responses in WT and Mda5-/- macrophages. Transfection of M. tuberculosis RNA strongly induced proinflammatory cytokine production in WT macrophages, which was abrogated in Mda5-/- macrophages. M. tuberculosis infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFN-γ-primed Mda5-/- macrophages effectively contained intracellular M. tuberculosis proliferation to a markedly greater degree than WT macrophages. Further comparisons of WT versus Mda5-/- macrophages revealed that during M. tuberculosis infection MDA5 contributed to IL-1ß production and inflammasome activation and that loss of MDA5 led to a substantial increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M. tuberculosis bacillary burden. These data reveal that loss of MDA5 is host protective during M. tuberculosis infection in vitro and in vivo, suggesting that M. tuberculosis exploits MDA5 to subvert immune containment.

Intravenous BCG vaccination reduces SARS-CoV-2 severity and promotes extensive reprogramming of lung immune cells.

Bacillus Calmette-Guérin (BCG) confers heterologous immune protection against viral infections and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here, we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model. BCG vaccination conferred a modest reduction on lung SCV2 viral load, bronchopneumonia scores, and weight loss, accompanied by a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. BCG uniquely recruited immunoglobulin-producing plasma cells to the lung suggesting accelerated local antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, with a transcriptional shift away from exhaustion markers and toward antigen presentation and repair. Similarly, BCG enhanced recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, that show reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Adjunctive Integrated Stress Response Inhibition Accelerates Tuberculosis Clearance in Mice.

Despite numerous advances in tuberculosis (TB) drug development, long treatment durations have led to the emergence of multidrug resistance, which poses a major hurdle to global TB control. Shortening treatment time therefore remains a top priority. Host-directed therapies that promote bacterial clearance and/or lung health may improve the efficacy and treatment duration of tuberculosis antibiotics. We recently discovered that inhibition of the integrated stress response, which is abnormally activated in tuberculosis and associated with necrotic granuloma formation, reduced bacterial numbers and lung inflammation in mice. Here, we evaluated the impact of the integrated stress response (ISR) inhibitor ISRIB, administered as an adjunct to standard tuberculosis antibiotics, on bacterial clearance, relapse, and lung pathology in a mouse model of tuberculosis. Throughout the course of treatment, ISRIB robustly lowered bacterial burdens compared to the burdens with standard TB therapy alone and accelerated the time to sterility in mice, as demonstrated by significantly reduced relapse rates after 4 months of treatment. In addition, mice receiving adjunctive ISRIB tended to have reduced lung necrosis and inflammation. Together, our findings identify the ISR pathway as a promising therapeutic target with the potential to shorten TB treatment durations and improve lung health. IMPORTANCE Necrosis of lung lesions is a hallmark of tuberculosis (TB) that promotes bacterial growth, dissemination, and transmission. This process is driven by the persistent hyperactivation of the integrated stress response (ISR) pathway. Here, we show that adjunctive ISR inhibition during standard antibiotic therapy accelerates bacterial clearance and reduces immunopathology in a clinically relevant mouse model of TB, suggesting that host-directed therapies that de-escalate these pathological stress responses may shorten TB treatment durations. Our findings present an important conceptual advance toward overcoming the challenge of improving TB therapy and lowering the global burden of disease.

Improved bladder cancer antitumor efficacy with a recombinant BCG that releases a STING agonist.

Despite the introduction of several new agents for the treatment of bladder cancer (BC), intravesical BCG remains a first line agent for the management of non-muscle invasive bladder cancer. In this study we evaluated the antitumor efficacy in animal models of BC of a recombinant BCG known as BCG-disA-OE that releases the small molecule STING agonist c-di-AMP. We found that compared to wild-type BCG (BCG-WT), in both the orthotopic, carcinogen-induced rat MNU model and the heterotopic syngeneic mouse MB-49 model BCG-disA-OE afforded improved antitumor efficacy. A mouse safety evaluation further revealed that BCG-disA-OE proliferated to lesser degree than BCG-WT in BALB/c mice and displayed reduced lethality in SCID mice. To probe the mechanisms that may underlie these effects, we found that BCG-disA-OE was more potent than BCG-WT in eliciting IFN-ß release by exposed macrophages, in reprogramming myeloid cell subsets towards an M1-like proinflammatory phenotypes, inducing epigenetic activation marks in proinflammatory cytokine promoters, and in shifting monocyte metabolomic profiles towards glycolysis. Many of the parameters elevated in cells exposed to BCG-disA-OE are associated with BCG-mediated trained innate immunity suggesting that STING agonist overexpression may enhance trained immunity. These results indicate that modifying BCG to release high levels of proinflammatory PAMP molecules such as the STING agonist c-di-AMP can enhance antitumor efficacy in bladder cancer.

Genetic and hormonal mechanisms underlying sex-specific immune responses in tuberculosis.

Tuberculosis (TB), the world's deadliest bacterial infection, afflicts more human males than females, with a male/female (M/F) ratio of 1.7. Sex disparities in TB prevalence, pathophysiology, and clinical manifestations are widely reported, but the underlying biological mechanisms remain largely undefined. This review assesses epidemiological data on sex disparity in TB, as well as possible underlying hormonal and genetic mechanisms that might differentially modulate innate and adaptive immune responses in males and females, leading to sex differences in disease susceptibility. We consider whether this sex disparity can be extended to the efficacy of vaccines and discuss novel animal models which may offer mechanistic insights. A better understanding of the biological factors underpinning sex-related immune responses in TB may enable sex-specific personalized therapies for TB.

Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

Recombinant BCGs for tuberculosis and bladder cancer.

Bacillus Calmette-Guérin (BCG) vaccine is an attenuated live strain of Mycobacterium bovis. It may be the most widely used vaccine in human history and is the only licensed human tuberculosis (TB) vaccine available. Despite its excellent safety history, a century of use in global vaccination programs, and its significant contribution to reducing TB mortality among children, the efficacy of BCG continues to be disputed due to its incomplete protection against pulmonary TB in adults. Still vaccines offer the best chance to contain the ongoing spread of multi-drug resistance TB and disease dissemination. The development of improved vaccines against TB therefore remains a high global priority. Interestingly, recent studies indicate that genetically modified BCG, or administration of existing BCG through alternate routes, or revaccination, offers improved protection, suggesting that BCG is well poised to make a comeback. Intravesical BCG is also the only approved microbial immunotherapy for any form of cancer, and is the first-line therapy for treatment-naïve non-muscle invasive bladder cancer (NMBIC), which represents a majority of the new bladder cancer cases diagnosed. However, almost a third of patients with NMIBC are either BCG unresponsive or have tumor recurrence, leading to a higher risk of disease progression. With very few advances in intravesical therapy over the past two decades for early-stage disease, and a limited pipeline of therapeutics in Phase 3 or late Phase 2 development, there is a major unmet need for improved intravesical therapies for NMIBC. Indeed, genetically modified candidate BCG vaccines engineered to express molecules that confer stronger protection against pulmonary TB or induce potent anti-tumor immunity in NMIBC have shown promise in both pre-clinical and clinical settings. This review discusses the development of second generation, genetically modified BCG candidates as TB vaccines and as anti-tumor adjuvant therapy for NMIBC.

Genetics of human susceptibility to active and latent tuberculosis: present knowledge and future perspectives.

Tuberculosis is an ancient human disease, estimated to have originated and evolved over thousands of years alongside modern human populations. Despite considerable advances in disease control, tuberculosis remains one of the world's deadliest communicable diseases with 10 million incident cases and 1·8 million deaths in 2015 alone based on the annual WHO report, due to inadequate health service resources in less-developed regions of the world, and exacerbated by the HIV/AIDS pandemic and emergence of multidrug-resistant strains of Mycobacterium tuberculosis. Recent findings from studies of tuberculosis infection and of patients with Mendelian predisposition to severe tuberculosis have started to reveal human loci influencing tuberculosis outcomes. In this Review, we assess the current understanding of the contribution of host genetics to disease susceptibility and to drug treatment. Despite remarkable progress in technology, only a few associated genetic variants have so far been identified, strongly indicating the need for larger global studies that investigate both common and under-represented rare variants to develop new approaches to combat the disease. Pharmacogenomic discoveries are also likely to lead to more efficient drug design and development, and ultimately safer and more effective therapies for tuberculosis.

Mechanisms of HIV-1 Control.

PURPOSE OF REVIEW: HIV-1 infection is of global importance, and still incurs substantial morbidity and mortality. Although major pharmacologic advances over the past two decades have resulted in remarkable HIV-1 control, a cure is still forthcoming. One approach to a cure is to exploit natural mechanisms by which the host restricts HIV-1. Herein, we review past and recent discoveries of HIV-1 restriction factors, a diverse set of host proteins that limit HIV-1 replication at multiple levels, including entry, reverse transcription, integration, translation of viral proteins, and packaging and release of virions. RECENT FINDINGS: Recent studies of intracellular HIV-1 restriction have offered unique molecular insights into HIV-1 replication and biology. Studies have revealed insights of how restriction factors drive HIV-1 evolution. Although HIV-1 restriction factors only partially control the virus, their importance is underscored by their effect on HIV-1 evolution and adaptation. The list of host restriction factors that control HIV-1 infection is likely to expand with future discoveries. A deeper understanding of the molecular mechanisms of regulation by these factors will uncover new targets for therapeutic control of HIV-1 infection.

Intracellular HIV-1 RNA and CD4+ T-cell activation in patients starting antiretrovirals.

OBJECTIVE: To assess if the reduction in HIV-1 RNA in CD4 T cells is correlated with the persistence of immune activation following early antiretroviral therapy (ART). DESIGN: Clinical trial (NCT01285050). METHODS: Next-generation sequencing was used to study total RNA from activated CD4 T cells (CD38 and human leukocyte antigen - antigen D related (HLA-DR) expressing) collected from 19 treatment-naïve HIV-1/hepatitis C virus-infected patients before and early after ART initiation (≥12 weeks after plasma HIV-1 RNA <50 copies/ml). To validate comparisons, pre and post-ART measures were adjusted for input RNA and overall read number. RESULTS: As expected, ART use was associated with a median [interquartile range (IQR)] 4.3% (2.2-8.3) reduction in the proportion of activated CD4 T cells (P = 0.0008). Whereas in those activated CD4 T cells no consistent differences in overall gene expression were detected, interferon-stimulated gene expression declined (P < 2 × 10). Pre-ART, sorted activated CD4 T cells contained a median (IQR) of 959 (252-1614) HIV-1 reads/10 reads compared with 72 (55-152) HIV-1 reads/10 reads after at least 12 weeks of suppressive ART (P = 8 × 10). The decrease in HIV-1 reads in activated CD4 T cells was associated with the change in plasma HIV-1 RNA levels (r = 0.77, P = 2 × 10) and the change in the proportion of activated CD4 T cells (r = 0.70, P = 0.0016). CONCLUSION: Months of ART led to a marked decrease in cell-associated HIV-1 RNA and interferon-stimulated genes expression in activated CD4 T cells that were strongly associated with the reduction in the proportion of activated CD4 T cells.

Stool microbiome reveals diverse bacterial ureases as confounders of oral urea breath testing for Helicobacter pylori and Mycobacterium tuberculosis in Bamako, Mali.

Detection of bacterial urease activity has been utilized successfully to diagnose Helicobacter pylori (H. pylori). While Mycobacterium tuberculosis (M. tuberculosis) also possesses an active urease, it is unknown whether detection of mycobacterial urease activity by oral urease breath test (UBT) can be exploited as a rapid point of care biomarker for tuberculosis (TB) in humans. We enrolled 34 individuals newly diagnosed with pulmonary TB and 46 healthy subjects in Bamako, Mali and performed oral UBT, mycobacterial sputum culture and H. pylori testing. Oral UBT had a sensitivity and specificity (95% CI) of 70% (46-88%) and 11% (3-26%), respectively, to diagnose culture-confirmed M. tuberculosis disease among patients without H. pylori, and 100% sensitivity (69-100%) and 11% specificity (3-26%) to diagnose H. pylori among patients without pulmonary TB. Stool microbiome analysis of controls without TB or H. pylori but with positive oral UBT detected high levels of non-H. pylori urease producing organisms, which likely explains the low specificity of oral UBT in this setting and in other reports of oral UBT studies in Africa.

The calcium-binding proteins S100A8 and S100A9 initiate the early inflammatory program in injured peripheral nerves.

To shed light on the early immune response processes in severed peripheral nerves, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P, regenerating) and distal (D, degenerating) nerve stumps on day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, IL, TNF, MHC, immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase, tissue inhibitor of metalloproteinase, and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerves. S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that, following peripheral nerve injury, an immediate acute immune response occurs both distal and proximal to the lesion site and that the rapid transcriptional activation of the S100a8 and S100a9 genes results in S100A8/A9 hetero- and homodimers, which stimulate the release of chemokines and cytokines by activated Schwann cells and generate the initial chemotactic gradient that guides the transmigration of hematogenous immune cells into the injured nerve.

N-glycosylation deficiency reduces ICAM-1 induction and impairs inflammatory response.

Congenital disorders of glycosylation (CDGs) result from mutations in various N-glycosylation genes. The most common type, phosphomannomutase-2 (PMM2)-CDG (CDG-Ia), is due to deficient PMM2 (Man-6-P → Man-1-P). Many patients die from recurrent infections, but the mechanism is unknown. We found that glycosylation-deficient patient fibroblasts have less intercellular adhesion molecule-1 (ICAM-1), and because of its role in innate immune response, we hypothesized that its reduction might help explain recurrent infections in CDG patients. We, therefore, studied mice with mutations in Mpi encoding phosphomannose isomerase (Fru-6-P → Man-6-P), the cause of human MPI-CDG. We challenged MPI-deficient mice with an intraperitoneal injection of zymosan to induce an inflammatory response and found decreased neutrophil extravasation compared with control mice. Immunohistochemistry of mesenteries showed attenuated neutrophil egress, presumably due to poor ICAM-1 response to acute peritonitis. Since phosphomannose isomerase (MPI)-CDG patients and their cells improve glycosylation when given mannose, we provided MPI-deficient mice with mannose-supplemented water for 7 days. This restored ICAM-1 expression on mesenteric endothelial cells and enhanced transendothelial migration of neutrophils during acute inflammation. Attenuated inflammatory response in glycosylation-deficient mice may result from a failure to increase ICAM-1 on the vascular endothelial surface and may help explain recurrent infections in patients.

Proinflammatory S100A12 can activate human monocytes via Toll-like receptor 4.

RATIONALE: S100A12 is overexpressed during inflammation and is a marker of inflammatory disease. Furthermore, it has been ascribed to the group of damage-associated molecular pattern molecules that promote inflammation. However, the exact role of human S100A12 during early steps of immune activation and sepsis is only partially described thus far. OBJECTIVES: We analyzed the activation of human monocytes by granulocyte-derived S100A12 as a key function of early inflammatory processes and the development of sepsis. METHODS: Circulating S100A12 was determined in patients with sepsis and in healthy subjects with experimental endotoxemia. The release of human S100A12 from granulocytes as well as the promotion of inflammation by activation of human monocytes after specific receptor interaction was investigated by a series of in vitro experiments. MEASUREMENTS AND MAIN RESULTS: S100A12 rises during sepsis, and its expression and release from granulocytes is rapidly induced in vitro and in vivo by inflammatory challenge. A global gene expression analysis of S100A12-activated monocytes revealed that human S100A12 induces inflammatory gene expression. These effects are triggered by an interaction of S100A12 with Toll-like receptor 4 (TLR4). Blocking S100A12 binding to TLR4 on monocytes or TLR4 expressing cell lines (HEK-TCM) abrogates the respective inflammatory signal. On the contrary, blocking S100A12 binding to its second proposed receptor (receptor for advanced glycation end products [RAGE]) has no significant effect on inflammatory signaling in monocytes and RAGE-expressing HEK293 cells. CONCLUSIONS: Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis.

S100A8 and S100A9: new insights into their roles in malignancy.

Recent studies have highlighted key roles played by non-neoplastic host cells of the tumor microenvironment, and by secreted factors from tumor and host cells, in promoting malignancy. In this regard, damage-associated molecular pattern (DAMP) molecules such as S100A8 and S100A9, with well-known functions in inflammation, have been increasingly recognized not only as markers, but also as new candidates with important roles in modulating tumor growth and metastasis. This review focuses on our current understanding of the pro- and anti-tumorigenic functions of S100A8 and S100A9. Elucidating molecular pathways mediated by these proteins promises to provide potential novel targets for the development of cancer therapeutics and to establish valid biomarkers to identify early stages of tumor progression.

S100A8/A9 activate key genes and pathways in colon tumor progression.

The tumor microenvironment plays an important role in modulating tumor progression. Earlier, we showed that S100A8/A9 proteins secreted by myeloid-derived suppressor cells (MDSC) present within tumors and metastatic sites promote an autocrine pathway for accumulation of MDSC. In a mouse model of colitis-associated colon cancer, we also showed that S100A8/A9-positive cells accumulate in all regions of dysplasia and adenoma. Here we present evidence that S100A8/A9 interact with RAGE and carboxylated glycans on colon tumor cells and promote activation of MAPK and NF-κB signaling pathways. Comparison of gene expression profiles of S100A8/A9-activated colon tumor cells versus unactivated cells led us to identify a small cohort of genes upregulated in activated cells, including Cxcl1, Ccl5 and Ccl7, Slc39a10, Lcn2, Zc3h12a, Enpp2, and other genes, whose products promote leukocyte recruitment, angiogenesis, tumor migration, wound healing, and formation of premetastatic niches in distal metastatic organs. Consistent with this observation, in murine colon tumor models we found that chemokines were upregulated in tumors, and elevated in sera of tumor-bearing wild-type mice. Mice lacking S100A9 showed significantly reduced tumor incidence, growth and metastasis, reduced chemokine levels, and reduced infiltration of CD11b(+)Gr1(+) cells within tumors and premetastatic organs. Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis.