ASSOCIATION OF GENERATION TIME WITH ANTI-TUBERCULAR DRUG(S) RESISTANCE PATTERN OF MYCOBACTERIUM TUBERCULOSIS ISOLATES AMONG TREATMENT FAILURE PULMONARY TUBERCULOSIS PATIENTS

ABSTRACTObjective: The emergence of drug resistance has complicated tuberculosis (TB) scenario and is associated to treatment failure. The causative agent,Mycobacterium tuberculosis is usually slow growing and has been implicated as a contributing factor for drug tolerance and development of resistantstrains. On the other hand, if rapidly growing bacilli, with shorter generation time emerge, mutations may lead to the development of drug resistance.From the hypothesis, this study was aimed to explore the whether there is any association between the generation time of Mycobacteria with theirdistinct drug resistant pattern.Methods: In-vitro generation time was determined from 77 mycobacterial isolates with varied drug resistance pattern, i.e. rifampicin resistant (RIFR),isoniazid resistant, multi-drug resistant (MDR), the sensitive clinical strains along with reference strains. The minimal inhibitory concentration wasalso determined for the respective resistant groups.Results: Among the individual group of clinical isolates, there was a significant negative association between generation time and drug resistancepattern of RIFR isolates.Conclusion: Keeping the current upsurge of the MDR-TB epidemic in India and the influence of generation time on dosing schedule and treatmentstrategy, necessary customization of dosing and therapeutic planning seemed urgent to minimize the operational and clinical potential for developmentof drug resistance among treatment failure pulmonary TB patients in this country.Keywords: Mycobacterium tuberculosis, Generation time, Multi-drug resistant, Treatment failure

Immune Subversion by <i>Mycobacterium tuberculosis</i> through CCR5 Mediated Signaling: Involvement of IL-10

<div><p>Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During <i>Mycobacterium tuberculosis</i> infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that <i>Mycobacterium</i> infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, <i>in vivo</i> CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen.</p></div

IL-10 augments the CCR5 expression in <i>H37Rv</i> infected macrophages via involving STAT3.

<p>Bone marrow derived macrophages (2×10⁶cells/ml) were pretreated with either anti IL-10 Ab (10 ug/ml) or with control siRNA and STAT3-specific siRNA and then infected with <i>Mycobacterium tuberculosis H37Rv</i> (MOI = 1∶10). Changes in messenger RNA (mRNA) expression of CCR5 and GAPDH were determined by semi quantitative RT-PCR (A). In a separate set, the pretreated and infected macrophages were lysed and subjected to Western blot with anti-CCR5 antibody as described in Materials and Methods (B). Infected macrophages were analyzed by flow cytometry for CCR5 (PE) expression as described in figure legend 1 (C). Data represented here are from one of three independent experiments, all of which yielded similar results. Murine macrophages (1×10⁶cells/ml) were treated with anti IL-10 Ab for 1 h and then subsequently followed by <i>Mycobacterium</i> tuberculosis infection for 45 min. After 45 min of incubation, ChIP assays were conducted as described in Materials and Methods. Immunoprecipitations were performed using Abs specific to acetylated H3 (IP acetyl-H3) (D) or STAT-3 (E), and conventional RT-PCR was performed using primers specific to the CCR5 promoter. Data represented here are from one of three independent experiments, all of which yielded similar results.</p

CCR5 silencing enhances pro inflammatory cytokine production and MHC-II expression in <i>H37Rv</i> infected C57BL/6 mice.

<p>C57BL/6 mice were transfected with CCR5 shRNA or control shRNA as described in materials and methods section prior to <i>Mycobacterium</i> infection. After 28 days of infection, the lung homogenates were assayed for the cytokine levels by ELISA as described in Methods (A–E). Data represented as means ± SD for 5 animals per group. ***<i>P</i><.001 and **<i>P</i><.05 for the comparison with infected mice. Total lung RNA was extracted and Changes in messenger RNA (mRNA) expression of IL-10, TGF-β, TNF-α, IL-12, IFN-γ and GAPDH were determined by semi quantitative RT-PCR (F). Data represented here are from one of three independent experiments, all of which yielded similar results. In separate experimental set, the lung homogenates were analyzed by flow cytometry for MHC-II (PE) expression as described in figure legend 1(G). Data represented here are from one of three independent experiments, all of which yielded similar results.</p

Effect of CCR5 on the survival of <i>Mycobacterium tuberculosis.</i>

<p>C57BL/6 mice were transfected with CCR5 shRNA or control shRNA as described in materials and methods section prior to <i>Mycobacterium</i> infection. After 28 days of infection, the lungs and spleens were lysed. The respective lysates were serially diluted and plated on Middle brook 7 H10 with Oleic acid-ADC in triplicate. Data are represented as log₁₀CFU/organ as mean ± SD. In a separate set, the transfected and infected mice were sacrificed and then the CCR5 expression in infected macrophages were analysed by semi quantitative RT-PCR (C) to validate the specific activity of shRNA mediated knockdown. Data represented here are from one of three independent experiments, all of which yielded similar results.</p

List of Primers.

<p>List of Primers.</p

The down-regulation of MHC-II expression in <i>H37Rv</i> infected macrophages was due to the CCR5 dependent IL-10 activation.

<p>Murine macrophages (2×10⁶cells/ml) were stimulated with IFN-γ (2 ng/ml) and then pretreated with either control siRNA or CCR5-specific siRNA for 24 h and 10 ug/ml anti IL-10 Ab for 1 h followed by <i>Mycobacterium</i> tuberculosis infection for 24 h. Infected macrophages were analyzed by flow cytometry for MHC-II (PE) expression as described in material method (A). Data represented here are from one of three independent experiments, all of which yielded similar results.</p

<i>Mycobacterium tuberculosis H37Rv</i> induces the enhancement of CCR5 expression in host macrophages.

<p>Bone marrow derived macrophages (2×10⁶) were cultured and then infected with <i>Mycobacterium tuberculosis H37Rv</i> [Multiplicity of Infection (MOI) = 1∶10] for different time points. Changes in messenger RNA (mRNA) expression of CCR5 and GAPDH were determined by semi quantitative RT-PCR (A). In a separate set, the infected macrophages were lysed and subjected to Western blot with anti-CCR5 antibody as described in Materials and Methods (B). Infected macrophages were analyzed by flow cytometry for CCR5 (PE) expression as described in material method (C). In a separate set, macrophages were treated with either CCR5 siRNA or Control siRNA or Lyn inhibitor PP2 and then infected as described above and cell lysates were subjected to western blot with either anti-Lyn antibody (D) or anti-ERK antibody (E). Data represented here are from one of three independent experiments, all of which yielded similar results.</p

<i>Mycobacterium tuberculosis H37Rv</i> infection disrupts the proinflammatory function of CCR5 in macrophages to produce high level of IL-10.

<p>Bone marrow derived macrophages (2×10⁶ cells/ml) were either treated with CCR5 siRNA or Control siRNA followed by <i>Mycobacterium tuberculosis H37Rv</i> (MOI = 1∶10) infection for 24 h and assayed for the levels of IL-10 (A), TGF-β (B), TNF-α (C), IL-12 (D) and IFN-γ (E) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means ± standard deviations of values from triplicate experiments that yielded similar observations. ***<i>P</i><.001 and **<i>P</i><.05 compared to that of the infected macrophages. In a separate set of experiment, siRNA pre treated macrophages were infected with <i>H37Rv</i> for 3 h. The extracellular bacteria were removed from the culture plate and the macrophages were incubated for another 3 hrs. RNA was isolated and semi quantitative RT-PCR analyses for TNF-α, IL-12, IFN-γ, IL-10, TGF-β and GAPDH were done. Data represented here are from one of three independent experiments, all of which yielded similar results (F). In separate experimental sets, Murine macrophages were pretreated with either control siRNA, CCR5-specific siRNA for 24 h or with Lyn inhibitor PP2, ERK inhibitor PD for 2 h, washed and then infected with <i>Mycobacterium</i> tuberculosis and changes in messenger RNA (mRNA) expression of IL-10 and GAPDH were determined by semi quantitative RT-PCR (G). Murine macrophages were transfected with siRNA and infected as mentioned above for 24 h and assayed for the levels of IL-10 (H) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. ***<i>P</i><.001 and **<i>P</i><.05 compared to that of the control siRNA treated infected macrophages. Murine macrophages (1×10⁶cells/ml) were treated as described previously and then subsequently followed by <i>Mycobacterium</i> tuberculosis infection for 45 min. After 45 min of incubation, ChIP assays were conducted as described in Materials and Methods. Immunoprecipitations were performed using Abs specific to phosphorylated H3 (IP phospho-H3) (I) or acetylated H3 (IP acetyl-H3) (J), and conventional RT-PCR was performed using primers specific to the IL-10 promoter. Data represented here are from one of three independent experiments, all of which yielded similar results.</p

Association between patient characteristics and drug resistance patterns among PTB patients in tertiary care hospitals of Kolkata, India, 2010–2013 (N = 350).

<p>SES = Socio-economic status; LIG = Low-income group; MIG = Middle-income group; HIG = High-income group; UOR = Unadjusted Odds ratio; AOR = Adjusted Odds ratio; OR = Odds ratio; CI = Confidence interval; <i>p</i> = <i>p</i> value.</p><p>“–” refers to cells for which due to inadequate number of observation valid statistical results (OR, CI, <i>p</i> value) could not be determined</p><p>Association between patient characteristics and drug resistance patterns among PTB patients in tertiary care hospitals of Kolkata, India, 2010–2013 (N = 350).</p