In this study, we present data suggesting that exposure to TB generates a distinct DNAm signature in pulmonary immune cells. The signature was found not only in those with active or latent TB infection, but also in individuals who are exposed but IGRA-negative. The finding that healthy, TB-exposed individuals also carry the signature opens the possibility that the epigenetic alterations reflect a host-beneficial reprogramming of the immune mechanisms rather than being induced by M. tuberculosis as a step to evade the immune defense. This notion is supported by our observation that the DMGs identified in the present study strongly overlapped with the epigenetic alterations identified in the in vitro BCG-trained macrophages, and the previously reported DNAm changes induced during BCG vaccination, which correlated with increased anti-mycobacterial capacity of macrophages4. In addition, we demonstrate that the GO data derived from our dataset display a strong overlap with data from a study on protective BCG vaccination in mice6.
BCG vaccination has convincingly been shown to induce heterologous immunity protecting against childhood mortality from other causes than TB20,21. Based on our finding that natural TB exposure and BCG vaccination trigger similar epigenetic changes we propose the hypothesis that a “beneficial exposure” to TB exists, which protects against other infections through heterologous immunity. Along the same line, it has been shown that a substantial fraction of individuals exposed to TB can be defined as ‘early clearers’, since they remain tuberculin skin test or IGRA negative22, suggesting effective eradication of the infection22. Identifying these early clearers and understanding the biology behind their resistance to TB infection could move the field forward towards novel strategies of TB prevention.
In concordance with the fact that macrophages constitute the main niche for mycobacterial replication, the strongest enrichment of DNAm changes was observed in the HLA-DR-positive cell population, which is dominated by alveolar macrophages. The pathways identified to be enriched in the HLA-DR-positive population have been described in the context of trained immunity, BCG exposure and TB. For example, activation of Hypoxia-Inducible Factor 1 α and glycolysis pathways (P00030 and P00024, respectively) are hallmarks of macrophages that have undergone the epigenetic changes reflective of trained immunity (reviewed in 23,24), which is induced in myeloid cells upon BCG-stimulation22,23. VEGF-release (P00056) by macrophages has been shown to recruit immune cells during granuloma formation25. Further, vitamin D has been shown to strengthen the anti-mycobacterial activity of macrophages11,26, and upregulation of components of the vitamin D pathway is linked to the production of anti-microbial peptides12, providing a possible effector mechanism for mycobacterial control. Recent literature on immune regulation through T cell-derived acetylcholine27,28 attributes relevance to the acetylcholine receptor pathway identified among the HLA-DR pathways.
Although macrophages and lymphocytes are not generally viewed as having many similarities, we found 34 of the identified pathways to overlap between HLA-DR- and CD3-positive cells. In data derived from the CD3 and PBMC populations, both of which represent lymphocytes, overlaps were identified for glycolysis, glutamate receptor and angiotensin II pathways. Interestingly, a metabolic shift towards increased glycolysis, representative of the Warburg effect, has been strongly associated with trained immunity23. However, the literature is dominated by the view that this event takes place in trained myeloid cells, while we identified this circuit in CD3 cells (T lymphocytes) and not in the HLA-DR cells (mainly macrophages). The glutamate receptor is widely expressed on immune cells and has been described as having an important regulatory role in T cells, which can also produce and release glutamate29. The role for angiotensin II pathway in TB remains elusive, while Angiotensin II Converting Enzyme 2 has been in the spotlight due to fact that the SARS-CoV2 virus utilizes it as a receptor for entry into host cells30. In the PBMC population, which over all showed a weaker epigenetic response, we found the interferon-γ signaling pathway, which has a well-established role in anti-mycobacterial defense (reviewed in 31), among the reprogramed pathways. Finally, several studies have ascribed Wnt pathways immunomodulating functions and induction during M. tuberculosis infection (reviewed in 14) and M1 and M2 macrophages express distinct patterns of Wnt ligands.
Taken together, we present data supportive of mycobacteria exposure-induced DNAm changes that correlate with previous findings from studies on BCG vaccination including TB protection and trained immunity.