A Blunted GPR183/Oxysterol Axis During Dysglycemia Results in Delayed Recruitment of Macrophages to the Lung During Mycobacterium tuberculosis Infection.

BACKGROUND: We previously reported that reduced GPR183 expression in blood from tuberculosis (TB) patients with diabetes is associated with more severe TB. METHODS: To further elucidate the role of GPR183 and its oxysterol ligands in the lung, we studied dysglycemic mice infected with Mycobacterium tuberculosis (Mtb). RESULTS: We found upregulation of the oxysterol-producing enzymes CH25H and CYP7B1 and increased concentrations of 25-hydroxycholesterol upon Mtb infection in the lungs of mice. This was associated with increased expression of GPR183 indicative of oxysterol-mediated recruitment of GPR183-expressing immune cells to the lung. CYP7B1 was predominantly expressed by macrophages in TB granulomas. CYP7B1 expression was significantly blunted in lungs from dysglycemic animals, which coincided with delayed macrophage infiltration. GPR183-deficient mice similarly had reduced macrophage recruitment during early infection. CONCLUSIONS: Taken together, we demonstrate a requirement of the GPR183/oxysterol axis for positioning of macrophages to the site of infection and add an explanation to more severe TB in diabetes patients.

Pre-Diabetes Increases Tuberculosis Disease Severity, While High Body Fat Without Impaired Glucose Tolerance Is Protective.

Type 2 diabetes (T2D) is a well-known risk factor for tuberculosis (TB), but little is known about pre-diabetes and the relative contribution of impaired glucose tolerance vs. obesity towards susceptibility to TB. Here, we developed a preclinical model of pre-diabetes and TB. Mice fed a high fat diet (HFD) for 12 weeks presented with impaired glucose tolerance and hyperinsulinemia compared to mice fed normal chow diet (NCD). Infection with M. tuberculosis (Mtb) H37Rv after the onset of dysglycemia was associated with significantly increased lung pathology, lower concentrations of TNF-α, IFN-γ, IFN-ß and IL-10 and a trend towards higher bacterial burden at 3 weeks post infection. To determine whether the increased susceptibility of pre-diabetic mice to TB is reversible and is associated with dysglycemia or increased body fat mass, we performed a diet reversal experiment. Pre-diabetic mice were fed a NCD for 10 additional weeks (HFD/NCD) at which point glucose tolerance was restored, but body fat mass remained higher compared to control mice that consumed NCD throughout the entire experiment (NCD/NCD). Upon Mtb infection HFD/NCD mice had significantly lower bacterial burden compared to NCD/NCD mice and this was accompanied by restored IFN-γ responses. Our findings demonstrate that pre-diabetes increases susceptibility to TB, but a high body mass index without dysglycemia is protective. This murine model offers the opportunity to further study the underlying immunological, metabolic and endocrine mechanisms of this association.

GPR183 Regulates Interferons, Autophagy, and Bacterial Growth During Mycobacterium tuberculosis Infection and Is Associated With TB Disease Severity.

Oxidized cholesterols have emerged as important signaling molecules of immune function, but little is known about the role of these oxysterols during mycobacterial infections. We found that expression of the oxysterol-receptor GPR183 was reduced in blood from patients with tuberculosis (TB) and type 2 diabetes (T2D) compared to TB patients without T2D and was associated with TB disease severity on chest x-ray. GPR183 activation by 7α,25-dihydroxycholesterol (7α,25-OHC) reduced growth of Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis BCG in primary human monocytes, an effect abrogated by the GPR183 antagonist GSK682753. Growth inhibition was associated with reduced IFN-ß and IL-10 expression and enhanced autophagy. Mice lacking GPR183 had significantly increased lung Mtb burden and dysregulated IFNs during early infection. Together, our data demonstrate that GPR183 is an important regulator of intracellular mycobacterial growth and interferons during mycobacterial infection.

RNA-Seq Revealed Expression of Many Novel Genes Associated With Leishmania donovani Persistence and Clearance in the Host Macrophage.

Host- as well as parasite-specific factors are equally crucial in allowing either the Leishmania parasites to dominate, or host macrophages to resist infection. To identify such factors, we infected murine peritoneal macrophages with either the virulent (vAG83) or the non-virulent (nvAG83) parasites of L. donovani. Then, through dual RNA-seq, we simultaneously elucidated the transcriptomic changes occurring both in the host and the parasites. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed (DE) genes, we showed that the vAG83-infected macrophages exhibit biased anti-inflammatory responses compared to the macrophages infected with the nvAG83. Moreover, the vAG83-infected macrophages displayed suppression of many important cellular processes, including protein synthesis. Further, through protein-protein interaction study, we showed significant downregulation in the expression of many hubs and hub-bottleneck genes in macrophages infected with vAG83 as compared to nvAG83. Cell signaling study showed that these two parasites activated the MAPK and PI3K-AKT signaling pathways differentially in the host cells. Through gene ontology analyses of the parasite-specific genes, we discovered that the genes for virulent factors and parasite survival were significantly upregulated in the intracellular amastigotes of vAG83. In contrast, genes involved in the immune stimulations, and those involved in negative regulation of the cell cycle and transcriptional regulation, were upregulated in the nvAG83. Collectively, these results depicted a differential regulation in the host and the parasite-specific molecules during in vitro persistence and clearance of the parasites.

IL-22: An Underestimated Player in Natural Resistance to Tuberculosis?

Approximately 10% of individuals latently infected with Mycobacterium tuberculosis (Mtb) develop active tuberculosis (TB) during their lifetime. Although it is well recognized that T-helper 1 immune responses are crucial for containing latent TB infection, the full array of host factors conferring protective immunity from TB progression are not completely understood. IL-22 is produced by cells of the innate and adaptive immune system including innate lymphoid cells, and natural killer cells as well as T lymphocytes (Th1, Th17, and Th22) and binds to its cognate receptor, the IL-22R1, which is expressed on non-hematopoietic cells such as lung epithelial cells. However, recent studies suggest that Mtb induces expression of the IL-22R1 on infected macrophages and multiple studies have indicated a protective role of IL-22 in respiratory tract infections. Reduced concentrations of circulating IL-22 in active TB compared to latent TB and decreased percentages of Mtb-specific IL-22 producing T cells in TB patients compared to controls designate this cytokine as a key player in TB immunology. More recently, it has been shown that in type 2 diabetes (T2D) and TB co-morbidity serum IL-22 concentrations are further reduced compared to TB patients without co-morbidities. However, whether a causative link between low IL-22 and increased susceptibility to TB and disease severity of TB exists remains to be established. This review summarizes the contribution of IL-22, a potentially under-appreciated key player in natural resistance to TB, at the interface between the immune response to Mtb and the lung epithelium.

Genome Plasticity in Cultured Leishmania donovani: Comparison of Early and Late Passages.

Leishmania donovani possesses a complex heteroxenic life cycle where infective metacyclic promastigotes are pre-adapted to infect their host and cope up with intracellular stress. Exploiting the similarities between cultured and sandfly derived promastigotes, we used early and late passage cultured promastigotes to show specific changes at genome level which compromise pathogen fitness reflected in gene expression and infection studies. The pathogen loses virulence mostly via transcriptional and translational regulations and long-time cultivation makes them struggle to convert to virulent metacyclics. At the genomic level very subtle plasticity was observed between the early and the late passages mostly in defense-related, nutrient acquisition and signal transduction genes. Chromosome Copy number variation is seen in the early and late passages involving several genes that may be playing a role in pathogenicity. Our study highlights the importance of ABC transporters and calpain like cysteine proteases in parasite virulence in cultured promastigotes. Interestingly, these proteins are emerging as important patho-adaptive factors in clinical isolates of Leishmania. We found that the currently available genome of Leishmania in the NCBI database are from late passages. Our early passage genome can act as a reference for future studies on virulent isolates of Leishmania. The annotated leads from this study can be used for virulence surveillance and therapeutic studies in the Indian subcontinent.

Cationic liposomal sodium stibogluconate (SSG), a potent therapeutic tool for treatment of infection by SSG-sensitive and -resistant Leishmania donovani.

Pentavalent antimonials have been the first-line treatment for leishmaniasis for decades. However, the development of resistance to sodium stibogluconate (SSG) has limited its use, especially for treating visceral leishmaniasis (VL). The present work aims to optimize a cationic liposomal formulation of SSG for the treatment of both SSG-sensitive (AG83) and SSG-resistant (GE1F8R and CK1R) Leishmania donovani infections. Parasite killing was determined by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic counting of Giemsa-stained macrophages. Macrophage uptake studies were carried out by confocal microscopic imaging. Parasite-liposome interactions were visualized through transmission electron microscopy. Toxicity tests were performed using assay kits. Organ parasite burdens were determined by microscopic counting and limiting dilution assays. Cytokines were measured by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Although all cationic liposomes studied demonstrated leishmanicidal activity, phosphatidylcholine (PC)-dimethyldioctadecylammonium bromide (DDAB) vesicles were most effective, followed by PC-stearylamine (SA) liposomes. Since entrapment of SSG in PC-DDAB liposomes demonstrated enhanced ultrastructural alterations in promastigotes, PC-DDAB-SSG vesicles were further investigated in vitro and in vivo. PC-DDAB-SSG could effectively alleviate SSG-sensitive and SSG-resistant L. donovani infections in the liver, spleen, and bone marrow of BALB/c mice at a dose of SSG (3 mg/kg body weight) not reported previously. The parasiticidal activity of these vesicles was attributed to better interactions with the parasite membranes, resulting in direct killing, and generation of a strong host-protective environment, necessitating a very low dose of SSG for effective cures.

Therapy with sodium stibogluconate in stearylamine-bearing liposomes confers cure against SSG-resistant Leishmania donovani in BALB/c mice.

BACKGROUND: Resistance of Leishmania donovani to pentavalent antimonials, the first-line treatment of visceral leishmaniasis (VL), has become a critical issue worldwide. Second-line and new drugs are also not devoid of limitations. Suitable drug-delivery systems can improve the mode of administration and action of the existing antimonials, thus increasing their clinical life. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the efficacy of sodium stibogluconate (SSG) in phosphatidylcholine (PC)-stearylamine-bearing liposomes (PC-SA-SSG), PC-cholesterol liposomes (PC-Chol-SSG) and free amphotericin B (AmB) against SSG-resistant L. donovani strains in 8-wk infected BALB/c mice. Animals were sacrificed and parasites in liver, spleen and bone marrow were estimated 4-wk post-treatment by microscopic examination of stamp smears and limiting dilution assay. A set of PC-SA-SSG and AmB treated mice were further studied for protection against reinfection. Serum antibodies and cytokine profiles of ex-vivo cultured splenocytes were determined by ELISA. Uptake of free and liposomal SSG in intracellular amastigotes was determined by atomic absorption spectroscopy. Rhodamine 123 and 5-carboxyfluorescein, known substrates of Pgp and MRP transporter proteins, respectively, were used in free and liposomal forms for efflux studies to estimate intracellular drug retention. Unlike free and PC-Chol-SSG, PC-SA-SSG was effective in curing mice infected with two differentially originated SSG-unresponsive parasite strains at significantly higher levels than AmB. Successful therapy correlated with complete suppression of disease-promoting IL-10 and TGF-ß, upregulation of Th1 cytokines and expression of macrophage microbicidal NO. Cure due to elevated accumulation of SSG in intracellular parasites, irrespective of SSG-resistance, occurs as a result of increased drug retention and improved therapy when administered as PC-SA-SSG versus free SSG. CONCLUSIONS/SIGNIFICANCE: The design of this single-dose combination therapy with PC-SA-SSG for VL, having reduced toxicity and long-term efficacy, irrespective of SSG-sensitivity may prove promising, not only to overcome SSG-resistance in Leishmania, but also for drugs with similar resistance-related problems in other diseases.

Comparison of liposome based antigen delivery systems for protection against Leishmania donovani.

Liposomes have been widely exploited as antigen delivery systems for a variety of diseases including leishmaniasis. These vesicles can be prepared in various ways which may affect the immunogenicity of the encapsulated antigens. In this study we compared the vaccine potentiality of three cationic formulations with Leishmania donovani promastigote membrane antigens (LAg) and the best vesicle was evaluated for long-term protection against experimental visceral leishmaniasis. We immunized mice with LAg encapsulated in multilamellar vesicles (MLV), dehydration-rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and challenged them with parasites ten days after vaccination. LAg in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. Protection observed with antigen incorporated MLV or DRV was predominantly Th1 as evidenced by elicitation of significantly high DTH, IgG2a antibodies and IFN-gamma. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly. Field trials of L. donovani vaccine were unsatisfactory mainly due to lack of an appropriate adjuvant. Cationic MLV when used as adjuvant with protein antigens induced sustained Th1 immunity. Adjuvant potential of cationic MLV can be utilized to design subunit vaccines.