Investigation of minor groove binders (MGB), non-ionic surfactant vesicles (NIV) delivery systems and IL-4i1 as novel pathogen- and host-directed drug therapy for tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis is the leading infectious disease epidemic that claims over 1.6 million lives, while 10 million fell ill in 2017. South Africa is burdened with the third highest global incidences following India and China with high rates of co-infections with HIV and highest numbers of multi-drug resistant (MDR) and extremely resistant (XDR) TB per capita. The current treatment regimen is decades old and requires a prolonged period of 6 months. The lack of efficient TB therapy and the emergence of MDR and XDR TB, there is an urgent need to find new drug targets for TB therapy through understanding the complex host-pathogen interactions. This may then lead to pathogen, host-directed therapies (HDT) or adjunct therapies as well as the development of effective drugs and drug formulations for the treatment of TB. Here we aimed to investigate potential targets for pathogen-and host-directed therapies for TB. We screened the anti-mycobacterial activity of 172 minor groove binder (MGB) compounds that selectively bind to AT-rich regions of the minor groove of bacterial DNA with the helical structure matching that of DNA in Mtb culture. Of the 172 total compounds screened 17 hits were identified, of which 2, MGB 362 and MGB 364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19 μM, respectively, whilst being non-toxic. Encapsulation of MGBs into non- ionic surfactant vesicles (NIVs) demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB alone. Treatment with MGB 364 or MGB 364 formulation did not cause DNA damage in murine infected macrophages as displayed by low expression of γ-H2Ax compared to H2O2 and DMSO. Intranasal administration of MGB 364 and MGB-NIV 364 formulation showed one log reduction in bacterial burden with improved pathology and immune cytokine production when in formulation. However, intranasal administration of 10 mg/kg MGB 362 together with rifampicin had no effect on bacterial loads. In summary, the data demonstrate the potential of MGB as a novel class of drug/chemical entity in anti-TB therapy and NIVs as an effective delivery system in a novel anti-TB formulation. Using deep CAGE and small RNA (CHIP-seq) technologies, International Center for Genetic Engineering and Biotechnology’s Cytokines and Diseases lab in collaboration with the RIKEN Center for Integrative Medical Sciences (Yokohama, Japan) performed a novel transcriptomics study approach by conducting a genome-wide transcriptional analyses of RNA transcripts from classically activated macrophages (caMph) and alternatively activated macrophages (aaMph) during Mtb infection. We identified host target genes that may play a role in host immune subverting mechanism by Mtb to hide away from host effector functions providing a possible target for host-directed therapy for tuberculosis. It is postulated that Mtb modulates the transcriptional landscape of IL-4/IL13 alternatively activated macrophages (aaMph) to escape killing by reactive nitrogen intermediates (NO) and reactive oxygen species (ROS) functions by IFN-γ stimulated classically activated macrophages (caMph). Here we report on the immunoregulatory role of IL-4i1, a candidate gene that was upregulated in aaMph during Mtb infection. IL-4i1 is a secreted L-amino oxidase with antibacterial properties. The enzyme converts Phenylalanine (Phe) into phenylpyruvate releasing toxic products ammonia and hydrogen peroxide (H2O2) which in-turn cause immunosuppression of effector T-cells by directly inhibiting polarization, proliferation and function or by promoting the generation of Foxp3 T-regulatory cells. Thus suggesting that IL-4i1 is involved in immune-regulatory mechanisms and may be implicated in immune evasion mechanisms by the pathogen. Here we report on the role of IL-4i1 on tissue localized T-cell activation and proliferative status thus maintaining immune local immune homeostasis. Thus showing that the absence of IL-4i1 could cause autoimmunity. To determine the functional role of IL-4i1 during Mtb infection, IL-4i1 deficient mice and wild-type littermate controls were infected with H37Rv and hypervirulent HN878 Mtb strain. IL-4i1 deficient mice were highly resistant to both strains of Mtb at 12- and 21-days post-infection as denoted by significant reduction in bacterial loads, reduced inflammation, reduced tissue iNOS expression reduced recruitment of interstitial macrophages, pro-inflammatory cytokines showed a trend for reduction. Interestingly there was a significant increase in NO production in infected tissues. There was an increase in M1-like macrophages that correlated with increased pro-inflammatory cytokines and chemokines. These data suggested that IL-4i1 regulates macrophage-mediated inflammatory responses during acute Mtb infection thus showing potential as an immunomodulatory target for TB HDT therapy. The study thus provides a framework for new drug targets for the development of new effective drugs and vaccines for TB therapy

Effects of host sex and pregnancy on Trichinella Zimbabwensis infection in Sprague-Dawley rats and Balb C mice.

M. Sc. University of KwaZulu-Natal, Durban 2014.Trichinellosis is a zoonotic parasitic disease caused by nematode parasites of the genus Trichinella. Trichinella species infect a wide range of hosts including humans, domestic and wild animals. The main mode of human infection is through ingestion of raw or undercooked pork. The successful establishment and development of Trichinella parasite in a host is affected by various factors which include sex of host, environmental, immunological and hormonal. The objective of this study was to determine the effect of host sex and pregnancy on the establishment and development of Trichinella zimbabwensis in Sprague-Dawley rats and Balb C mice respectively. Rodents are the common reservoirs of Trichinella spp. in the domestic and sylvatic cycles and it was logical to determine the establishment and development of Trichinella zimbabwensis in Sprague-Dawley rats. The study on the effects of pregnancy and levels of progesterone and cortisol was done in Balb C mice and this was influenced by availability of mice in large quantities to conduct the experiments. Rats and mice are not widely different in their physiology and have been used interchangeably as host in previous Trichinella studies. Therefore it was important to use these animals as models for the study. In order to determine the effect of host sex on the establishment and development of T. zimbabwensis, 50 Sprague-Dawley rats were divided into two groups (25 males and 25 females) and orally infected with 7 Trichinella zimbabwensis muscle larvae per gram (LPG) of animal live weight. On days 5, 10, 15, 20 and 25 post-infection (PI), five animals from each group were sacrificed and the numbers of adult parasites in the intestine as well as larvae in muscles were determined. To determine the effect of host pregnancy, 90 female Balb C mice were divided into 3 groups of 30 mice each. Group 1 animals were orally infected with 50 LPG on day 0 of trial; group 2 animals were mated on day 0, but were not infected; group 3 animals were mated on day 0 and infected with 50 LPG on day 7 post-mating. On days 0, 7, 14, 21 and 28 PI for groups 1; days 0, 7, 14, 21 and 28 post-mating for group 2; and days 7, 14, 21, 28 and 35 post-mating for group 3, six animals from each group were sacrificed and the numbers of adult parasites in the intestines as well as larvae in the muscles were determined in infected groups. In addition, levels of the hormones progesterone and cortisol were measured in all groups at the same intervals. Results from the study showed a significantly higher number of Trichinella adults and larvae (P < 0.05) in male than in female Sprague-Dawley rats (four times higher adult worms and two times higher in muscle larvae in males than in females). On the other hand, pregnancy reduced the number of larvae establishing in muscles with progesterone levels significantly higher in pregnant than in non-pregnant Balb C mice (P < 0.05). This was attributed to the parasiticidal effect of progesterone against new-born larvae (NBL). This finding can be exploited when designing strategies to control and to treat the infection in rodents and humans. There were no significant differences in cortisol levels between pregnant and non-pregnant mice

Evaluation of Minor Groove Binders (MGBs) as novel anti-mycobacterial agents, and the effect of using non-ionic surfactant vesicles as a delivery system to improve their efficacy

Objectives: The slow development of major advances in drug discovery for the treatment of Mycobacterium tuberculosis (Mtb) infection have led to a compelling need for evaluation of more effective drug therapies against tuberculosis. New classes of drugs are constantly being evaluated for anti-mycobacterial activity with currently a very limited number of new drugs approved for TB treatment. Minor Groove Binders (MGBs) have previously revealed promising anti-microbial activity against various infectious agents; however have not yet been screened against Mtb. Methods: Mycobactericidal activity of MGB compounds against Mtb was determined using H37Rv-GFP microplate assay. MGB hits were screened for their intracellular mycobactericidal efficacy against clinical Beijing Mtb strain HN878 in bone marrow-derived macrophages using standard colony-forming unit counting. Cell viability was assessed by CellTiter-Blue assays. Selected MGB were encapsulated into non-ionic surfactant vesicles (NIVs) for drug delivery system evaluation. Results: H37Rv-GFP screening yielded a hitlist of 7 compounds at an MIC99 between 0.39 and 1.56 μM. MGB-362 and MGB-364 displayed intracellular mycobactericidal activity against Mtb HN878 at MIC50 of 4.09 μM and 4.19 μM respectively, whilst being non-toxic. Subsequent encapsulation into NIVs demonstrated a 1.6 and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared to MGB alone formulation Conclusions: MGBs anti-mycobacterial activities together with non-toxic properties indicate that MGB compounds constitute an important new class of drug/chemical entity, which holds promise in future anti-TB therapy. Furthermore, NIVs ability to better deliver entrapped MGB compounds to an intracellular Mtb infection has provided merit for further preclinical evaluation

Host regulation of liver fibroproliferative pathology during experimental schistosomiasis via interleukin-4 receptor alpha

<div><p>Interleukin-4 receptor (IL-4Rα) is critical for the initiation of type-2 immune responses and implicated in the pathogenesis of experimental schistosomiasis. IL-4Rα mediated type-2 responses are critical for the control of pathology during acute schistosomiasis. However, type-2 responses tightly associate with fibrogranulomatous inflammation that drives host pathology during chronic schistosomiasis. To address such controversy on the role of IL-4Rα, we generated a novel inducible IL-4Rα-deficient mouse model that allows for temporal knockdown of <i>il-4rα</i> gene after oral administration of Tamoxifen. Interrupting IL-4Rα mediated signaling during the acute phase impaired the development of protective type-2 immune responses, leading to rapid weight loss and premature death, confirming a protective role of IL-4Rα during acute schistosomiasis. Conversely, IL-4Rα removal at the chronic phase of schistosomiasis ameliorated the pathological fibro-granulomatous pathology and reversed liver scarification without affecting the host fitness. This amelioration of the morbidity was accompanied by a reduced Th2 response and increased frequencies of FoxP3⁺ Tregs and CD1d^hiCD5⁺ Bregs. Collectively, these data demonstrate that IL-4Rα mediated signaling has two opposing functions during experimental schistosomiasis depending on the stage of advancement of the disease and indicate that interrupting IL-4Rα mediated signaling is a viable therapeutic strategy to ameliorate liver fibroproliferative pathology in diseases like chronic schistosomiasis.</p></div

Generation and genotypic characterization of the RosaCre^-/+ IL-4Rα^-/lox deletable mouse model.

<p><b>A.</b> IL-4Rα^-/- C57BL/6 mice were intercrossed with RosaCre expressing and IL-4Rα^Lox/Lox mice to generate RosaCre^-/+ IL-4Rα^-/lox (i.e. iCre^-/+ IL-4Rα^-/lox mice). <b>B.</b> Schematic of the expected <i>il-4r</i> gene loci in iCre^-/+ IL-4Rα^-/lox mice fed or not with Tamoxifen. <b>C.</b> Experimental design for assessing the inducible deletion of <i>il-4rα</i> in Tamoxifen-fed iCre^-/+ IL-4Rα^-/lox mice. <b>D.</b> Short-term effect of Tamoxifen gavage on body weight. <b>E.</b> Genotyping of iCre^-/+ IL-4Rα^-/lox mice. The <i>creer</i>^<i>T2</i> specific amplicon is 300bp, <i>loxp</i> is 450bp. In wild type mice, the <i>il-4rα</i> amplicon is 600bp and 471bp in mice with a deleted <i>il-4rα</i> gene. <b>F.</b> Semi-quantitative PCR analyses of Exon 8 (only deleted in mice with an activated CreER^T2) vs. Exon 5 (present in all mice) between iCre^-/+ IL-4Rα^{-/lox mice} fed either with oil or Tamoxifen. (<b>G, H).</b> Quantitative real-time PCR of exon 8 over exon 5 from the <i>il-4rα</i> gene. Genomic DNA was extracted from liver tissue (<b>G</b>) or splenocytes (<b>H</b>) and exon 8 from <i>il-4rα</i> was quantified by qPCR and normalized to exon 5. Each experiment was conducted at least twice with 3–4 mice per group. Data are expressed as mean ± SD; NS = p > 0.05; * = p < 0.05; ** = p < 0.01; *** =, p < 0.001; **** = p < 0.0001.</p

Interleukin-4 receptor alpha is still required after Th2 polarization for the maintenance and the recall of protective immunity to Nematode infection

<div><p>There is currently no vaccine against parasitic nematodes and the knowledge on the mechanisms by which protective immunity against this class of parasites is achieved is continuously expanding. Nematode parasites trigger a host protective type 2 immune response via interleukin-4 receptor alpha (IL-4Rα). Despite this central role, it is not known whether IL-4Rα has a role in maintaining host type 2 immune responses following polarization. To determine the role of IL-4Rα after polarization, we used a recently established strain of rosaCreER^T2-/+IL-4Rα^-/Lox mice where <i>il4rα</i> gene deletion can be temporally controlled. We show that sustained expression of IL-4Rα is required for the maintenance of type 2 immune responses and protective immunity following interruption after polarization with <i>Nippostrongylus brasiliensis</i> primary infection. Moreover, we show by temporal deletion of IL-4Rα prior to secondary infection with <i>N</i>. <i>brasiliensis</i> that signaling via this receptor drives more efficient recall of type 2 immune responses and clearance of the parasites. Together, this study demonstrates that sustained IL-4Rα mediated signaling is required for the maintenance of anti-nematode type 2 immune responses, describing a novel function for IL-4Rα that is distinct from its role in immune polarization.</p></div

Kinetics of interruption of IL-4Rα expression following tamoxifen administration to i^Cre-/+IL-4Rα^-/Lox mice.

<p><b>A.</b> Experimental design. <b>B.</b> Gating strategy for Blood CD19⁺CD4^- B cells and CD19^-CD4⁺ T-cells. <b>C.</b> Formula for the relative quantification of the levels of IL-4Rα expression on blood B cells. A level of 100% is defined here as the difference between the average IL-4Rα GMFI on cells from control mice (IL-4Rα^-/Lox) and that of the average IL-4Rα expression on cells from IL-4Rα deficient mice (IL-4Rα^-/-). The relative expression of IL-4Rα of i^Cre-/+IL-4Rα^-/Lox mice fed with tamoxifen as in <b>A</b> is summarized in <b>D</b> for Blood CD19⁺ B cells and E for Blood CD4⁺ T-cells. The experiment was conducted with 3–4 mice per group. Data are expressed as mean ± SD.</p

Different phenotypes after knocking down IL-4Rα at different phases of experimental schistosomiasis.

<p><b>A.</b> Experimental design. <b>B.</b> Survival curve representing the cumulative profile from 2 different infections (n = 5–10 mice).</p

Sustained IL-4Rα mediated signaling is critical for the anti-parasitic type 2 immune responses during primary <i>N</i>. <i>brasiliensis</i> infection.

<p>Mice were infected with 500 L3 <i>N</i>. <i>brasiliensis</i>, fed with tamoxifen once daily from day 5 to day 8 post infection and killed 9 days post-infection. <b>A.</b> Schematic showing the experimental design. <b>B.</b> IL-4Rα expression on CD3⁺CD4⁺ T-cells from MLN of <i>N</i>. <i>brasiliensis</i>-infected mice. <b>C.</b> Cytokine production detected by ELISA in the supernatant of restimulated MLN cells. <b>D.</b> Percentages of cytokine-producing CD4⁺ T-cells after stimulation with PMA/Ionomycin/Monensin cocktail. <b>E.</b> Percentages of transcription factor-expressing CD3⁺CD4⁺ T-cells (<i>ex-vivo</i>) summarized in (<b>F</b>)<b>. G.</b> Total serum IgE in <i>N</i>. <i>brasiliensis-infected</i> mice. <b>H.</b> NbAg-specific serum type 2 antibodies. <b>I.</b> Eosinophil (SiglecF^hi) numbers per MLN. <b>J.</b> Worm counts per gut. Each experiment was conducted at least twice with 4–12 mice per group. Data are expressed as mean ± SD; NS = p > 0.05; * = p < 0.05; ** = p < 0.01; *** =, p < 0.001; **** = p < 0.0001.</p

Immunological and histopathological profile of <i>S</i>. <i>mansoni-</i>infected mice after knocking down IL-4Rα 2 weeks post-infection.

<p><b>A.</b> Experimental design. <b>B.</b> Representative plot of MLN cytokine-producing CD3⁺CD4⁺ T cell frequencies after stimulation with PMA/Ionomycin/Monensin cocktail. Summaries of IL-4-producing (<b>C</b>) and IFNγ-producing (<b>D</b>) CD3⁺CD4⁺ T cell frequencies from 2 independent experiments conducted with 3–8 mice are shown. <b>E.</b> Cytokine release detected by ELISA in the supernatant of anti-CD3 stimulated MLN cells. <b>F.</b> Total seric IgE in <i>S</i>. <i>mansoni</i>-infected mice. SEA-specific seric IgG1 (<b>G</b>) and IgG2a (<b>H</b>) isotype antibodies. <b>I.</b> Liver Egg burden. <b>J.</b> Formalin-fixed Hematoxylin/Eosin-stained sections of liver tissue from infected animals for morphological analyses (displayed here at 100X). <b>K.</b> Area sizes of egg-surrounding granuloma are computed. <b>L.</b> Formalin-fixed CAB-stained sections of liver tissue from infected animals for collagen detection (displayed here at 100X). <b>M.</b> Hydroxyproline content measured by colorimetry is displayed as a measure of tissue collagen content. Liver (<b>N</b>) and Spleen (<b>O</b>) weights. <b>P.</b> Body weight change over time following <i>S</i>. <i>mansoni</i> infection. <b>Q.</b> Survival curve following <i>S</i>. <i>mansoni</i> infection. Each experiment was conducted at least twice with 5–10 mice per group. Data are expressed as mean ± SD; NS = p > 0.05; * = p < 0.05; ** = p < 0.01; *** =, p < 0.001; **** = p < 0.0001.</p