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Reply to Vani et al

  1. Gucheng Zeng1,
  2. Dan Huang1,
  3. Crystal Y. Chen1,
  4. Ling Shen2,
  5. Richard R. Wang1 and
  6. Zheng W. Chen1
  1. 1 Department of Microbiology and Immunology, Center for Primate Biomedical Research, University of Illinois College of Medicine, Chicago, Illinois
  2. 2 Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts
  1. Reprints or correspondence: Zheng W. Chen, MD, PhD, 909 S. Wolcott Ave., University of Illinois College of Medicine, Chicago, IL 60612 (zchen{at}uic.edu).

To the Editor—We appreciate that Vani et al have made use of our observations to study cellular responses and cytokines of B cells in patients with tuberculosis. They found that B cells from tuberculosis patients produced an increased amount of the inflammatory cytokines interleukin (IL)-8 and IL-6, with some changes in the levels of IL-1, IL-12 p70, IL-10, or tumor necrosis factor, suggesting that severe tuberculosis in human induced the up-regulation of B cell inflammatory cytokines, but low levels of antigen-specific B cell responses [1]. We agree that their observation is relevant to our large-scale gene-expression studies. However, we note that their experiments were designed using an in vitro stimulation approach, rather than direct in vivo investigation.

We have recently shown that severe tuberculosis can induce unbalanced up-regulation of immune gene networks and overexpression of IL-22 and other inflammatory cytokine transcripts in lymphocytes from the lungs of macaques [2]. Because IL-22 transcripts were overexpressed in lymphocytes from the lungs of macaques with severe tuberculosis, we asked whether IL-22 protein could be directly detected in tuberculosis granuloma lesions in lung tissues. To address this question, we visualized IL-22 protein in cells in the tuberculosis granuloma tissues of M. tuberculosis-infected macaques, using con-focal microscopy imaging. Frozen sections of lung ∼5 µm thick were prepared, as described elsewhere [3], from optimal cutting temperature compound-embedded lung tissue collected from M. tuberculosis-infected Chinese rhesus macaques, which were infected with 500 colony-forming units of bacilli by bronchoscope-guided inoculation into the right caudal lobe and euthanized at day 63 after infection [3]. Tissue sections were first incubated with polyclonal rabbit anti-human IL-22 (N-terminus; Capralogics) and monoclonal mouse anti-human CD3 (F7.2.38; Dako), and then incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin (Ig) G (Biolegend) and Cy3-conjugated goat anti-mouse IgG (Biolegend). The tissue sections were then mounted on slides using fluorescence mounting medium with 4′,6-diamidino-2-phenylindole for confocal microscope imaging (LSM 650; Zeiss).

As shown in the figure, IL-22 protein was clearly detectable in CD3+ T cells in the tuberculosis granuloma lesions from M. tuberculosis-infected macaques. In contrast, no IL-22 was detected in lung tissue sections from uninfected, healthy macaques vaccinated with bacille Calmette-Guérin 4 years before. The direct visualization of IL-22 in T cells in tuberculosis granuloma tissues by confocal microscopy is in agreement with our previous work, which showed that severe tuberculosis induced 220-fold up-regulation of the gene encoding IL-22 in the lungs of macaques [2]. It is technically noteworthy that most cytokines, such as interferon-γ, are hardly detected or visualized by direct staining without in vitro antigen restimulation. It is also important to mention that IL-22 may play a dual role in host immune responses to tuberculosis, depending on host and microbial conditions. “Physical-level” IL-22 production (i.e., not overproduction) during low-level mycobacterial infection might facilitate potential antimicrobial responses [4, 5]. On the other hand, overproduction of IL-22 due to high tuberculosis burdens might lead to a more inflammatory outcome, a consequence seen in the setting of dermal inflammation [6]. Although the immunopathogenesis of tuberculosis is complex, our findings make it possible ultimately to elucidate the role of IL-22 in M. tuberculosis infection.

Figure
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Figure

Representative confocal microscopic images (63× numerical aperture) demonstrate that interleukin (IL)-22 protein was readily detectable in CD3+ T cells in the right caudal lung lobes (infection site) containing tuberculosis granuloma in Mycobacterium tuberculosis-infected macaques (“M.tb-infected,” lower panels). In contrast, no IL-22 was detected in the lung tissue sections from uninfected, healthy macaques vaccinated with bacille Calmette-Guérin 4 years before (“Uninfected,” upper panels). The white arrowheads in the merged figure in the lower row indicate the IL-22+CD3+ T cells. At least 20 imaging views were obtained of 6 different tissue sections collected from 3 monkeys for semiquantitative analyses. No IL-22 staining with very low background-level fluorescence was observed when isotype control immunoglobulin G or antibodies not reactive with tissue-sectioned IL-22 were used for similar immune staining (data not shown).

 
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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: National Institutes of Health (R01 grants HL64560 and RR13601 to Z.W.C.).

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  1. J Infect Dis. (2009) 200 (3): 482-483. doi: 10.1086/599844
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