Immune Responses of HIV-1 Tat Transgenic Mice to Mycobacterium Tuberculosis W-Beijing SA161

Abstract

Background: Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis.

Results: Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijing SA161. Survival assays demonstrate accelerated mortality rates of the Tat transgenic mice compared to non-transgenics. Tat transgenic mice also showed poorly organized lung granulomata-like lesions. Analysis of the host immune response using quantitative RT-PCR, flow cytometry for surface markers, and intracellular cytokine staining showed increased expression of pro-inflammatory cytokines in the lungs, increased numbers of cells expressing ICAM1, increased numbers of CD4+CD25+Foxp3+ T regulatory cells, and IL-4 producing CD4+ T cells in the Tat transgenics compared to infected non-tg mice.

Conclusions: Our data show quantitative differences in the inflammatory response to the SA161 clinical isolate of M. tuberculosis W-Beijing between Tat transgenic and non-transgenic mice, suggesting Tat contributes to the pathogenesis of tuberculosis.

Keywords: HIV-1; Mycobacterium tuberculosis W-Beijing; Tat; immune responses..

Fig. (1)

Tat-tg mice infected with M. tuberculosis W-Beijing SA161 show decreased survival compared to non-tg mice, but similar lung bacterial burden. (a) Survival of non-tg and Tat-tg mice was monitored after a low dose aerosol infection with M. tuberculosis W-Beijing SA161 (blue closed circles and red open squares, respectively) or the control laboratory strain H37Rv (orange hash marks and grey open circles, respectively). n=5 mice per group. The arrows indicate day 65 post infection. (b) Bacterial counts from non-tg (blue bars) and Tat-tg mice (red bars) infected with W-Beijing SA161 at the indicated times post infection. Results are expressed as the average Log₁₀ CFUe (±SE) of the bacterial load in each experimental group (n=4).

Fig. (2)

Increased lung pathology in Tat–tg mice infected with M. tuberculosis W-Beijing SA161 compared to non-tg control mice. (a) Representative photomicrograph of H&E-stained lungs from an uninfected Tat-tg mouse. Total magnification a=100x. (b-e) Lung sections from all 4 mice per group infected with a low dose aerosol of W-Beijing SA161 M. tuberculosis 30 days post infection were examined and a representative photomicrographs of H&E-stained lungs from a non-tg (b) and (c) or a Tat-tg mouse (d and e) are shown. Total magnification b, d=20x; c, e=200x. (f) H&E sections were analyzed; total lung area from all 4 mice were measured and compared to areas affected by lesions. The percent lung involvement is reported; SA161-infected Tat-tg mice (red bar), non-tg mice (blue bar).

Fig. (3)

Increased pro-inflammatory cytokine expression in the lungs of Tat-tg mice infected with M. tuberculosis W-Beijing SA161. At various time points post infection (Day 7, 15, and 30), TNFα (a) and IFN-γ (b) mRNA levels were quantified from the lungs of mice. In both cases, the non-tg mice are represented by the blue closed circles and Tat-tg mice are shown as red open squares. (n=4 mice per group). In the absence of bacterial infection, TNFα or IFN-γ expression levels are indicated by the “uninfected” samples shown in both a and b.

Fig. (4)

Higher numbers of both CD4+CD25+ T cells that express Foxp3+ and CD4+ T cells that express IL-4 in Tat-tg mice infected with M. tuberculosis W-Beijing SA161. Flow cytometry was used to quantify the number of cells in the lung. CD4+ T cells were first identified by gating on SSC vs CD4+ T cells. From these, cells that also express CD25+ and Foxp3+ 30 days post infection were gated as shown in the representative dot plot (a). (b) The number of CD4+CD25+Foxp3+ T cells in the lungs of Tat-tg (red open squares) and non-tg (blue closed circles) mice at various time points after infection are reported. Results are expressed as the average number of CD4+CD25+Foxp3 (***, p<0.0002) +/- SEM. (n=4). (c) CD4+ T cells that also express IL-4 were gated; a representative blot at day 30 is shown. (d) The number of CD4+IL-4+ T cells in the lungs of non-tg and Tat-tg mice after infection were quantified at various time points. Results are expressed as the average number of CD4+IL-4 (***, p<0.0005) cells in each group, +/- SEM. (n=4).

Fig. (5)

Increased ICAM1 expression in Tat-tg mice infected with M. tuberculosis W-Beijing SA161. (a) Lung expression of ICAM1 by SA161-infected Tat-tg (red open squares) and non-tg (blue closed circles) mice was measured by qRT-PCR (**, p =0.006). Samples labeled “uninfected” represent mice that were not infected with mycobacteria. (b) ICAM1+ CD8+ T cells were also gated; a representative blot at day 30 is shown. (c) The total number of ICAM1+ CD8+ T cells in the lungs of Tat-tg (red) and non-tg mice (blue) after infection are shown (n=4). Results are expressed as the average number of ICAM1+ CD8+ T cells, +/- SEM. Samples labeled “uninfected” represent mice that were not infected with mycobacteria.