Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis

Abstract

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a novel glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomics analysis of lungs from JHU083-treated Mtb-infected mice revealed reduced glutamine levels, citrulline accumulation suggesting elevated NOS activity, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. When tested in an immunocompromised mouse model of Mtb infection, JHU083 lost its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

Keywords: Glutamine inhibition; Host-directed therapy; Immunotherapy; TB drug; Tuberculosis.

Fig 1.. DON has direct anti-mycobacterial activity in vitro.

(A) Chemical structures of the prodrug JHU083 and the active drug DON. Esterases and peptidases ubiquitously present in the blood and tissues convert JHU083 into DON. (B) Chemical structure of MSO. (C) Table showing the MIC values of these drugs against Mtb H37Rv strain determined using Alamar Blue Assay. (D) IFNƳ-activated BMDM were infected with Mtb H37Rv at an MOI of 2. They were then treated with 1X and 10X MIC concentrations of DON. Isoniazid (INH) was used as the positive control. The cells were lysed at indicated time points and plated on 7H11 selection plates. The assay was performed as described in the “Methods”. Data were plotted as Mean ±SEM. Statistical significance was calculated using a two-tailed student t-test considering unequal distribution. *<0.05, **<0.01, ***<0.001. All the experiments were performed in triplicates.

Fig 2.. JHU083 administration prevents Mtb proliferation in vivo.

(A) Schematic of the in vivo challenge experiment. 129S2 or C3HeB/FeJ mice (n=4–5) were aerosol infected with ~200–300 CFU of Mtb H37Rv. The mice were treated with JHU083 or RIF via oral route one day after infection. 1 mg/Kg JHU083 was given daily for the first five days, and then the dose was reduced to 0.3 mg/Kg daily (M-F). (B) The mice were sacrificed at day 0, weeks 2 and 5 post-infection/treatment. The lungs were harvested, homogenized, serially diluted, and plated on 7H11 selection plates. After 21–25 days, the colonies were counted, and counts were transformed into log₁₀ values and plotted. (C) The graph shows the percent increase in the body weight of the C3HeB/FeJ mice compared to day 0 with and without JHU083 treatment. (D) The survival of control and treated 129S2 mice during the experiment. (E) Gross lung weight of 129S2 mice at weeks 2 and 5 post infection/treatment. (F) The histopathology of the lungs isolated from C3HeB/FeJ mice infected with Mtb H37Rv at week 4 post-infection/treatment. The lungs were formalin fixed, sectioned, and stained with hematoxylin and eosin. (G) Quantitation of the lung granuloma areas in the C3HeB/FeJ mice infected with Mtb H37Rv at week 4 post-infection/treatment (n=6). Both total granuloma (GA) and lung (LA) areas were measured using ImageScope software (Leica). The percent granuloma (%GA) was calculated using the formula (%GA = (GA × 100)/LA). Data were plotted as Mean ± SEM. Statistical significance was calculated using a two-tailed student t-test considering unequal distribution. For survival study, the drug dosing was discontinued after 6 weeks. For survival curve, log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests were used and yielded similar p-value. *<0.05, **<0.01, ***<0.001, ****<0.0001. All the experiments were repeated at least two-times.

Fig 3.. JHU083 loses therapeutic efficacy in Mtb-infected immunocompromised mice.

(A) Schematic of the in vivo experiment. B, Balb/c SCID mice (n=5) were aerosol infected with ~40 CFU of Mtb H37Rv. The mice were then treated with JHU083 or RIF via oral route one day after infection. 1 mg/Kg JHU083 was given daily for the first 5 days and then the dose was reduced to 0.3 mg/Kg daily (M-F) and given for 4 more weeks. (B) The mice were sacrificed at week 5 post-infection/treatment. The lungs were harvested, homogenized, serially diluted, and plated on 7H11 selection plates. After 21–25 days, the colonies were counted, and counts were transformed into log₁₀ values and plotted. (C) The graph shows the effect of both the PBS and Rif controls, and the drug treatment groups on the body weights of SCID mice during the experiment. (D) The graph shows the survival of SCID mice during the experiment. Data were plotted as Mean ± SEM. Statistical significance was calculated using a two-tailed student t-test considering unequal distribution. For survival curve, log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests were used and yielded similar p-value. *<0.05, **<0.01, ***<0.001, ****<0.0001. The experiment was repeated two times.

Fig 4.. JHU083 administration initiates earlier recruitment of T-cells in the lungs.

As described in Fig 2A, Mtb-infected 129S2 mice were treated with JHU083 and RIF daily starting day 1 post infection. The mice were sacrificed on weeks 2 and week 5, and the lungs were harvested. Single cell suspensions of the lungs from all three groups were stained with appropriate antibodies and analyzed using multicolor-flow cytometry (n=4–5). We found differences in the (A) CD4⁺ T-cell frequency, (B) TNFα expression upon activated CD4⁺ T-cells, (C) IFNƳ expression upon activated CD4⁺ T-cells, (D) IL-10 expression upon activated CD4⁺ T-cells, (E) Naïve CD4⁺ T-cells, (F) Follicular helper T-cells, (G) CD62L expression on CD8⁺ T-cells, (H) BCL6 expression on CD8⁺ T-cells, (I) Klrg1 expression on CD4⁺ T-cells, (J) Klrg1 expression on CD8⁺ T-cells, (K) frequency of Mature B-cells, (L) frequency of memory B-cells. The X-axis stands for the timepoint at which the lungs were harvested for the flow cytometry experiment. All T-cells data (A-J) came from the lungs harvested at week 2 (W2) after infection and treatment while B-cell data (K-L) was generated from lungs harvested at week 5 (W5) post- infection and treatment. Data were plotted as Mean ± SEM and are shown as the frequency of CD45⁺ population. gMFI stands for geometric mean fluorescence intensity and was used to define the expression of the individual markers upon the indicated cell types. Statistical significance was calculated using a two-tailed student t-test considering unequal distribution. *<0.05, **<0.01, ***<0.001, ****<0.0001. The experiment was repeated two times.

Fig 5.. JHU083 treatment causes infiltration of the proinflammatory myeloid cells in the lungs.

As described in Fig 2A, Mtb-infected 129S2 mice were treated with JHU083 and RIF every day starting day 1 post-infection. The mice were sacrificed on weeks 2 and week 5, and the lungs were harvested. Single cell suspension of the lungs from all three groups were stained with appropriate antibodies and analyzed using multicolor-flow cytometry (n=5). Details are provided in “Methods” section. We found differences in the (A) CD11b⁺ myeloid cells, (B) alveolar macrophages (AM), (C) CD86 expression upon AM, (D) CD206 expression upon AM, (E) Interstitial macrophages (IM), (F) IL-10 expression upon monocytic MDSCs (mMDSCs), (G) IL-10 expression upon granulocytic MDSCs (gMDSCs). The X-axis shows the timepoint at which the lungs were harvested for flow cytometry experiment. Data were plotted as Mean ± SEM and are shown as the frequency of CD45⁺ population. gMFI stands for geometric mean fluorescence intensity and was used to define the expression of the individual markers upon the indicated cell types. Statistical significance was calculated using a two-tailed student t-test considering unequal distribution. *<0.05, **<0.01. The experiment was repeated two times.

Fig 6.. JHU083 treatment drives metabolic reprogramming in the Mtb-infected lungs.

As described in Fig 2A, Mtb-infected 129S2 mice were treated with JHU083 and RIF every day starting day 1 post-infection. Mice were sacrificed at week 2, the lungs were harvested, and total metabolites were methanol extracted as described in “Methods”. The total metabolites were normalized to the tissue weight and then to the untreated controls. We detected changes in the level of (A) glutamine, (B) Citrulline, (C) 5-HydroxyIndole acetic acid (5-HIAA) and (D) Quinolinic acid. Schematic representation of the (E) arginine and (E) tryptophan metabolism listing the metabolic steps and the metabolites relevant to the study. Green and red arrows next to a metabolite indicate accumulation and depletion respectively in JHU083-treated group compared to untreated control. Abbreviations stands for; Arginine decarboxylase (ADC); L-arginine:glycine amidinotransferase (AGAT); Arginase (ARG); Guanidinoacetate N-methyltransferase (GAMT); Nitric oxide synthase (NOS); Ornithine decarboxylase (ODC); Ornithine decarboxylase (OTC); Dopa decarboxylase (DDC); 3-hydroxyanthranilate 3,4-dioxygenase (HAAO); Indoleamine 2,3-dioxygenase (IDO); Kynurenine 3Monooxygenase (KMO); Kynureninase (KYNU); Monoamine oxidase A/B (MAOA/B); Tryptophan 2,3dioxygenase (TDO2); Tryptophan hydroxylase (TPH1/2). Data were plotted as Mean ± SEM. Statistical significance was calculated using a two-tailed student t-test considering unequal distribution. *<0.05, **<0.01. The experiment was repeated two times.

Fig 7.. Model depicting the mechanism of action of JHU083.

Under glutamine-sufficient conditions, IL-10 produced by immunosuppressive myeloid cells (MDSCs) and quinolinic acid (a product of the Kynurenine pathway) inhibit T-cell proliferation ad functions, promoting infection and disease progression. Glutamine antagonist JHU083, decreases IL-10-producing MDSCs leading to a higher frequency of T-cells. Depletion of quinolinic acid and accumulation of citrulline further potentiates T-cell functions while preventing Mtb proliferation in macrophages. These immunometabolic changes then lead to disease regression and improved lung histology. Red lines represent host-deleterious process, while green lines are for host-protective processes.