SUMMARY Identification of unique leads represents a significant challenge in drug discovery. This hurdle is magnified in neglected diseases such as tuberculosis. We have leveraged public high-throughput screening (HTS) data, to experimentally validate virtual screening approach employing Bayesian models built with bioactivity information (single-event model) as well as bioactivity and cytotoxicity information (dual-event model). We virtually screen a commercial library and experimentally confirm actives with hit rates exceeding typical HTS results by 1-2 orders of magnitude. The first dual-event Bayesian model identified compounds with antitubercular whole-cell activity and low mammalian cell cytotoxicity from a published set of antimalarials. The most potent hit exhibits the in vitro activity and in vitro/in vivo safety profile of a drug lead. These Bayesian models offer significant economies in time and cost to drug discovery.

Nitric oxide (NO) produced by macrophages is toxic to host tissues and invading pathogens and its regulation is therefore essential to suppress host cytotoxicity. Macrophage arginase 1 (Arg1) inhibits the production of NO by competing with NO synthases for arginine, the common substrate of NO synthases and arginases. Two signal transduction pathways control the production of Arg1 in macrophages. First, a pathway dependent on the Toll-like receptor (TLR) adaptor protein myeloid differentiation marker 88 (MyD88) induces the expression of Arg1 in intracellular infections, whereas a second pathway, which is dependent on signal transducer and activator of transcription 6 (STAT6) is required for the expression of Arg1 in alternatively-activated macrophages. We found that mycobacteria-infected macrophages produced soluble factors, including interleukin-6 (IL-6), IL-10, and granulocyte colony-stimulating factor (G-CSF), that induced the expression of Arg1 in an autocrine-paracrine manner. We further established that Arg1 expression was controlled by the MyD88-dependent production of IL-6, IL-10, and G-CSF rather than by cell-intrinsic MyD88 signaling to Arg1. Our data reveal that the MyD88-dependent pathway that induces expression of Arg1 after infection by mycobacteria requires the activation of STAT3 and may result in the development of an immunosuppressive niche in granulomas because of the induced production of Arg1 in surrounding uninfected macrophages.* This manuscript has been accepted for publication in Science Signaling. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencesignaling.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the

Significance Failure of the host immune system to control infection with Mycobacterium tuberculosis is a major determinant of tuberculosis (TB) disease. In this work, we examined the role of macrophage migration inhibitory factor (MIF), a cytokine that is encoded in a functionally polymorphic locus in humans, in TB. We found genetic low expressers of MIF to be enriched in a population of patients with HIV and disseminated TB. From our work in cellular and mouse models, we propose a key mechanism by which MIF regulates bacterial recognition as the first step in triggering inflammatory pathways to enable mycobacterial control.

Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro-and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the leading infectious disease causes of morbidity and mortality worldwide. Though current antibiotic regimens can cure the disease, treatment requires at least six months of drug therapy. One reason for the long duration of therapy is that the currently available TB drugs were selected for their ability to kill replicating organisms and are less effective against subpopulations of non-replicating persistent bacilli. Evidence from in vitro models of Mtb growth and mouse infection studies suggests that host immunity may provide some of the environmental cues that drive Mtb towards non-replicating persistence. We hypothesized that selective modulation of the host immune response to modify the environmental pressure on the bacilli may result in better bacterial clearance during TB treatment. For this proof of principal study, we compared bacillary clearance from the lungs of Mtb-infected mice treated with the anti-TB drug isoniazid (INH) in the presence and absence of an immunomodulatory phosphodiesterase 4 inhibitor (PDE4i), CC-3052. The effects of CC-3052 on host global gene expression, induction of cytokines, and T cell activation in the lungs of infected mice were evaluated. We show that CC-3052 modulates the innate immune response without causing generalized immune suppression. Immune modulation combined with INH treatment improved bacillary clearance and resulted in smaller granulomas and less lung pathology, compared to treatment with INH alone. This novel strategy of combining anti-TB drugs with an immune modulating molecule, if applied appropriately to patients, may shorten the duration of TB treatment and improve clinical outcome.

Pyrazinamide (PZA) is one of the first line antibiotics used for the treatment of tuberculosis (TB). In the present study, we have used in vitro and in vivo systems to investigate whether PZA, in addition to its known anti-mycobacterial properties, modulate the host immune response during Mycobacterium tuberculosis (Mtb) infection. In vitro we have examined the effect of PZA on cytokine and chemokine release by Mtb-infected or Toll-like receptor (TLR) -stimulated primary human monocytes. In vivo, we have investigated at the transcriptional levels using genome-wide microarray gene expression analysis, whether PZA treatment of Mtb-infected mice alters the host immune response to Mtb infection in the lungs. Here, we report that PZA treatment of Mtb-infected human monocytes and mice significantly reduces the release of pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, TNF-α and MCP-1 at the protein and at the gene transcription levels, respectively. Data from microarray analysis also reveal that PZA treatment of Mtb-infected mice significantly alters the expression level of genes involved in the regulation of the pro-inflammatory mediators, lung inflammatory response and TLR signaling networks. Specifically, genes coding for adenylate cyclase and Peroxisome-Proliferator Activated Receptor (PPAR), molecules known for their anti-inflammatory effect, were found to be up-regulated in the lungs of PZA-treated Mtb-infected mice. Based on the microarray findings, we propose that PZA treatment modulates the host immune response to Mtb infection by reducing pro-inflammatory cytokine production, probably through PPAR- and NF-kB- dependent pathways. In addition, our results suggest that inclusion or exclusion of PZA in the TB treatment regimen could potentially affect the biomarker signature detected in the circulation of TB patients.

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