HLA‑DRB1*1501 and VDR polymorphisms and survival of Mycobacterium tuberculosis in human macrophages exposed to inhalable microparticles

Pharmacogenomics. 2013 Apr;14(5):531-40.

Singh A K, Abhimanyu, Yadav A B, Sharma S, Garg R, Bose M and Misra A

CSIR-Central Drug Research Institute, Lucknow 226001, India.

 

Abstract:

AIM:

We examined whether HLA-DRB1*1501 and four VDR SNPs influence the macrophage response to infection with Mycobacterium tuberculosis (Mtb) via innate immune mechanisms versus drug treatment or drug delivery mechanisms.

MATERIALS & METHODS:

Monocyte-derived macrophages from 24 healthy donors were infected with Mtb in vitro. Survival of intracellular bacilli and secretion of cytokines and nitric oxide by the infected cells were monitored with and without exposure to isoniazid and rifabutin.

RESULTS:

Haplotype analysis was conducted, and an arbitrary score of genetic ‘susceptibility’ (S) score ranging from -3 to +3 was assigned to donors based on the presence or absence of genetic markers. S scores correlated more strongly with Mtb survival (r = 0.68) than TNF and nitric oxide (NO; r = ∼0.01-0.11). A specific haplotype was significantly associated with decreased Mtb survival (p < 0.05), increased NO and decreased IL-10/IL-4. Macrophages with S scores ≥ 2 secreted significantly (p < 0.05) more IL-10 and IL-4, and less NO upon infection, and supported Mtb survival. Microparticulate drugs showed higher bactericidal activity than free drugs, irrespective of S score.

CONCLUSION:

S score predicts colonization of macrophages by Mtb, as does haplotype analysis. Drug-containing microparticles are superior to free drugs across diverse genetic backgrounds.

PMID: 23556450

 

Supplement:

Host susceptibility to infectious diseases, including tuberculosis (TB), is two pronged—with environmental and genetic components contributing to establishment and progression of infection. Appropriate nutrition, for example prevents and even reverses TB progression. Poor socio-economic status, overcrowding, etc are contributing factors promoting infection and disease progression. Environmental factors are not, however, sufficient to explain individual susceptibility. There is a genetic component of susceptibility to TB. In 1926, 251 babies were given a wrong TB vaccine. 77 babies died and 127 developed TB, but 47 remained asymptomatic. Susceptibility may result in part from variability of the genes controlling host immune responses. Several host genes are associated with susceptibility to Mtb and M. leprae. The human leucocyte antigen gene (HLA class-II complex) vitamin D receptor gene (VDR), natural resistance associated macrophage protein -1 gene (NRAMP-1), Interferon gamma and various other cytokine genes show appreciable association with TB.  HLA genes are highly polymorphic, suggesting a role of selection pressure against infectious agents.  The vitamin D receptor gene is a candidate for TB susceptibility. There are >490 single nucleotide polymorphisms (SNPs) in the VDR gene but four SNPs are the best studied: TaqI, BsmI, ApaI and FokI.

Recent work in our lab has proposed the use of inhalable microparticles containing a combination of anti-TB drugs for use as adjunct therapy in the treatment of pulmonary TB (Misra, Hickey et al. 2011) and demonstrated  high efficacy in animal experiments and cell lines (Sharma, Saxena et al. 2001; Muttil, Kaur et al. 2007; Verma, Kaur et al. 2008) , and also apparently “stimulate the phagocyte” as recommended by Koch a hundred years ago.

Since there is a great deal of genetic variation relevant to host defence responses of humans, it is important to investigate whether observations made using a single defined strain of Mtb (H37Rv) infecting macrophages of a single defined genotype (THP-1) would hold in the context of genetic variation observed in a sample of a human population. The present investigations were undertaken to evaluate host response and bacterial survival when primary macrophages derived from human volunteers were infected in vitro with Mtb H37Rv and treated with inhalable microparticles containing isoniazid (INH) and rifabutin (RFB) reported extensively from the lab.

Amit Singh-11

Figure1. (A). Macrophages of ~90% Individuals are classically Activated by Mtb. (B). Microparticles rescue alternatively-activated macrophages (re-drawn from Verma et al., 2011)

Volunteers were genotyped for VDR polymorphism and presence or absence of the HLA DRB1*1501 allele. Apart from the standard population genetics methodology of constructing a haplotype block, an arbitrary score of ‘susceptibility’ and ‘resistance’ to macrophage colonization by Mtb could be constructed.  Each genetic marker was assigned an arbitrary value of 1 on the ‘susceptibility’ or ‘resistance’ scale, as applicable. Thus, if 4 factors associated with ‘susceptibility’ and one associated with ‘resistance’ were present, S was read as 4-1=+3. Donor MDMs could be ranked in descending order of ‘susceptibility’ to infection. Further, volunteers were randomly selected from each score group and peripheral blood monocytes were differentiated to macrophages and infected and/or treated with microparticles containing drug or drugs in solution. NO production and cytokine secretion by infected/treated macrophages, and survival of intracellular bacteria were evaluated.

Amit Singh-2

Figure 2: PBMC-derived Macrophage from volunteers were infected in vitro with 10 MOI of Mtb H37Rv, Left untreated (black); treated with INH+RFB in MP (blue) or equivalent amounts of drugs in solution. CFU in cell lysate were estimated by plating 48-hr cell lysates on 7H10-OADC Agar.

S was consistent with haplotype. There was significant difference in Mtb survival between macrophages of the more susceptible haplotype i.e. T-b-a-F (or S score higher than 3) and less susceptible ones (t-B-A-F / S score<3) following infection and treatment. Drug-containing microparticles rather than drugs in solution significantly reduced CFU count in the more susceptible haplotype/higher S Score. Donors of less susceptible haplotype/ S Score produced higher level of NO following infection at 12 hrs and microparticles containing drug secreted more NO compared to soluble drugs at all times sampled. Donors with the higher susceptibility score/ haplotype had higher Th2 cytokine indices following infection and drug-containing microparticles performed better than soluble drugs in reducing the Th-2 cytokine index.

Despite limited sample size, results impart some valuable insights about the colonization of bacteria and its correlation with genetic susceptibility. Analysis of bacterial survival in the context of an arbitrary ‘Susceptibility Score’ and its substantiation by haplotype analysis suggests a possibility of evaluation of disease susceptibility and response to treatment, extending the range of applicability of S scores. A larger number of loci may need to be typed before arriving at a sufficiently comprehensive evaluation of macrophage susceptibility to sustainable colonisation by Mtb. Yet, the results demonstrate that microparticles are more efficacious than molecular solutions of anti-TB drugs at inhibiting intracellular Mtb survival in macrophages of diverse genotypes in the experimental conditions employed here.

 

References:

1. Misra, A., A. J. Hickey, et al. (2011). “Inhaled drug therapy for treatment of tuberculosis.” Tuberculosis (Edinb) 91(1): 71-81.

2. Muttil, P., J. Kaur, et al. (2007). “Inhalable microparticles containing large payload of anti-tuberculosis drugs.” Eur J Pharm Sci 32(2): 140-50.

3. Sharma, R., D. Saxena, et al. (2001). “Inhalable microparticles containing drug combinations to target alveolar macrophages for treatment of pulmonary tuberculosis.” Pharm Res 18(10): 1405-10.

4. Verma, R. K., J. Kaur, et al. (2008). “Intracellular time course, pharmacokinetics, and biodistribution of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to mice.” Antimicrob Agents Chemother 52(9): 3195-201.

5. Verma, R. K., A. K. Singh, et al. (2011). “Inhaled therapies for tuberculosis and the relevance of activation of lung macrophages by particulate drug-delivery systems.” Therapeutic Delivery  2(6): 753–768.

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