The Purinergic P2X7 Receptor Is Not Required for Control of Pulmonary Mycobacterium tuberculosis Infection

Control of M. tuberculosis after aerosol infection in P2X7^−/− mice.

P2X7^−/− mice backcrossed six generations to C57BL/6 (Pfizer) mice and wild-type C57BL/6 mice (Charles River) received low-dose (∼50 CFU) aerosol infection with virulent M. tuberculosis Erdman as previously described (14). At various time points postinfection, mice were sacrificed to determine bacillary burdens in the lungs and draining mediastinal lymph node (LN) (13). In two independent experiments, there were no differences in total lung CFU from 14 to 100 days postinfection between P2X7^−/− and control wild-type mice (Fig. 1A). A third experiment examined very early time points, and no difference in CFU in the lungs at day 5 or 10 was detected (data not shown). Bacillary burdens were also determined in the mediastinal LN in three experiments. There was no significant difference in numbers of M. tuberculosis CFU between the P2X7^−/− mice and wild-type controls (Fig. 1B). This suggests that the P2X7 receptor is not required for the control of pulmonary M. tuberculosis infection.

Mononuclear cells in naive and infected mice.

Initial experiments were performed to determine whether the baseline number and composition of mononuclear cells were similar in P2X7^−/− and control mice. There were no significant differences in absolute numbers of cells or CD4⁺-, CD8⁺-, CD11c⁺-, or CD11b⁺-expressing cells in lungs or spleen between naive P2X7^−/− and control mice (data not shown).

After aerosol M. tuberculosis infection, there were significantly higher total numbers of CD4⁺ T cells at weeks 4 (P = 0.05) and 11 (P = 0.005) in the lungs of P2X7^−/− mice (Fig. 2A). CD8⁺ T-cell numbers in the lungs were similar in both groups after infection (Fig. 2B). There were no statistically significant differences in the percentages of CD11c⁺ and CD11b⁺ cells in lungs and mediastinal LN after infection in two experiments (data not shown).

Several experiments were performed on infected P2X7^−/− and wild-type mice, using Annexin V and 7-aminoactinomycin D (7-AAD) staining, that did not show consistently different levels of apoptosis in either the T cells, dendritic cells (DC), or macrophages (data not shown).

Antigen-specific responses in lung and lymph node after infection.

After infection, T-cell responses were assessed using two methods. First, T-cell responses from lungs and mediastinal LN were analyzed by intracytoplasmic gamma interferon (IFN-γ) staining after anti-CD3 and anti-CD28 stimulation. We have shown previously that this correlates well with T-cell responses of previously activated CD44^high T cells (13). Total numbers of CD4⁺ T cells producing IFN-γ in the lungs of P2X7^−/− mice were significantly higher at weeks 4 (P = 0.02) and 11 (P = 0.02) after infection, with a trend also at weeks 6 and 14 (Fig. 2C). Similarly, total numbers of CD8⁺ T cells producing IFN-γ in the P2X7^−/− lungs were higher at weeks 4 (P = 0.02) and 11 (P = 0.007) (Fig. 2D).

To confirm that these responses were M. tuberculosis specific, IFN-γ enzyme-linked immunospot analysis was performed on the lungs and LN of infected animals in two separate experiments, using DC with and without M. tuberculosis infection to restimulate the T cells as previously described (7). In the lungs, there was no significant difference in M. tuberculosis-specific IFN-γ-secreting T cells between the groups (Fig. 2E). There were significantly higher numbers of M. tuberculosis-specific IFN-γ-secreting T cells in the LN of P2X7^−/− mice at 2 and 11 weeks postinfection (P < 0.05). Otherwise, there was a trend at all time points tested for higher M. tuberculosis-specific IFN-γ-secreting-T-cell frequencies in P2X7^−/− mice.

In vitro priming and antigen presentation.

We sought to determine a mechanism for the increased numbers of antigen-specific T cells in P2X7^−/− mice. Antigen presentation and priming functions were examined. Bone marrow-derived macrophages (BMM) were activated with IFN-γ and infected in vitro with M. tuberculosis. Antigen processing and presentation by BMM was determined using a M. tuberculosis antigen 85B-specific T-cell hybridoma (11). T-cell responses were identical after stimulation with either P2X7^−/− or wild-type BMM (Fig. 3A), demonstrating no difference in the intrinsic ability of P2X7^−/− macrophages to present M. tuberculosis antigens.

The ability of P2X7^−/− CD11c⁺ antigen-presenting cells (APC) to prime naive T cells was then determined. CD11c⁺ APC, primarily DC, were purified by immunomagnetic beads (Miltenyi) from spleens of naive P2X7^−/− and wild-type mice. T cells from naive ovalbumin (OVA)-specific T-cell transgenic mice (1) were incubated with OVA antigen and CD11c⁺ APC. An [³H]thymidine (ICN, Costa Mesa, CA) incorporation lymphoproliferation assay was performed. P2X7^−/− CD11c⁺ APC were similar to wild-type APC in their ability to prime naive T cells (Fig. 3B).

Overall, our studies demonstrate that the P2X7 receptor is not required in mice to contain M. tuberculosis infection. There was a modest but statistically significant increase in the number of IFN-γ-secreting CD4⁺ T cells in lungs of M. tuberculosis-infected P2X7^−/− mice. The etiology of this increase is not clear from our studies. It is not related to increased efficiency of P2X7^−/− APC in presenting antigen or in priming naive T cells. In addition, we were unable to detect consistent differences in apoptosis between the groups to account for the difference in T cells.

Polymorphisms in the human P2X7 gene have not been associated with increased risk of tuberculosis (8, 9, 12). Our previous in vitro work with human macrophages exposed to ATP from degranulating T cells or lysed bystander cells did not induce P2X7-mediated control of mycobacterial growth (2). These human data, along with the data presented in this in vivo P2X7^−/− mouse study, bring into question the importance of the P2X7 receptor in the control of mycobacterial disease. Perhaps the observation that high-dose extracellular ATP induces killing of mycobacteria through increases in phagolysomal fusion is just an interesting in vitro phenomenon that does not have a role in mycobacterial disease.