The main characteristics of MTB and MB pathogenicity primarily involved MTB and MB induced alterations in lipid metabolism to obtain nutrients for their development and colonization in macrophages. In response to this, macrophages could be polarized to M1 type to resist pathogen infection, mediated via activation of lipopolysaccharide (LPS) and Toll-like receptor (TLR) signaling 21. In the present study, it was observed that MTB induced more alterations in BAM lipid composition as compared to MB infection. Various lipids, especially glycerophospholipids, were found to be increased in BAM following MTB and MB infection, and thus it was concluded that glycerophospholipids could serve as clinical indicators for tuberculosis detection. Importantly, MTB also induced alterations in the contents of ceramide phosphocholines, FA glycosides 1‐alkyl,2‐acylgly, and N‐acylsphingosines, whereas MB only induced accumulation of 1‐alkyl,2‐acylglycerophosphoinositols. Furthermore, MTB dramatically invoked the expression of genes involved in various immune signaling‐ and lipid‐related pathways, including TNF signaling pathway, NOD‐like receptor signaling pathway, Toll‐like receptor signaling pathway, and fatty acid metabolism. However, MB attacks did not induce dramatic activation of signaling pathways or lipid‐related metabolism. In consensus with aforementioned metabolomics results, these phenomena suggested that MTB induced more alterations in BAM lipid metabolism. Therefore, the present study provided in‐depth/deep insights into different mechanisms of defense responses elicited in BAM against MTB and MB, which would contribute towards the detection and treatment of human and cattle tuberculosis.
Glycerophospholipids are known to play an important role in the maintenance of cell membrane stability and signal transduction in host cells 22. The results of the present study showed that MTB and MB attacks induced an increase in the contents of glycerophospholipids, suggesting that both these microbes might destroy macrophage membrane structure, while host macrophages could invoke intracellular lipid-related signal to activate defense responses. Besides this, glycerophospholipids are also known to play important roles in energy conversion, vesicle formation, and transmembrane transport 19. Thus, glycerophospholipids could serve as a nutrient for the host cell or MTB/MB. Additionally, polykelides, one of the upregulated categories, have been previously shown to exhibit anti‐bacterial and anti‐parasitic functions. Thus, an increase in these compounds would contribute towards macrophage defense against MTB/MB. Sterol lipids are important components of cell membrane structure, which are known to play a positive regulatory role in resisting pathogen infection 23. Consequently, an increase in these lipids would strengthen the host resistance towards MTB/MB attacks. Importantly, it has been previously reported that the levels of sphingolipids were increased in macrophages during MTB infection. Moreover, sphingolipids have been previously shown to be involved in host immune system, which could further induce inflammatory response in macrophages to strengthen host resistance. 24. It was previously reported that MTB infection primarily affected lipid metabolism of macrophages for successful development and colonization 25. The present data showed an increase in the content of glycerolipids in MTB/MB‐attacked macrophages. Similarly, it has been previously established that MTB induced accumulation of glycerolipids and liposomes as nutrient, for its development in macrophages 26. Meanwhile, MTB induced the accumulation of lipids mainly via alteration of glycolytic pathway to ketone body synthesis 27. Thus, disorder of lipid metabolism in macrophages would contribute towards tuberculosis occurrence 28. Additionally, MTB could further stimulate the accumulation of tumor necrosis factor‐α (TNF‐α) to suppress cholesterol metabolism in macrophages and increase lipid intake, resulting in the formation of FM 8. The present study reported dramatic activation of TNF‐α signaling pathway in MTB‐infected macrophages, which provided suitable explanation for the pathogenesis of MTB. Importantly, accumulation of lipids could further inhibit autophagy‐related pathways, resulting in the formation of FM, which weakened the phagocytosis of MTB/MB and inhibited their proliferation 29,30. The present study identified that increased lipids 1‐alkyl and 2‐acylglycerophosphoinositols, 1‐alkyl and 2‐acylglycerophosphates, C19 steroids, and diacylglycerophosphoinositols provided convenience for MB and MTB colonization in macrophages. In fact, these lipids could be used as clinical indicators for tuberculosis.
The phenomenon that more defense-related responses were activated during MTB‐attacks supported the current hypothesis that bovine macrophages were more sensitive to MTB than MB. Among these defense responses, various signaling‐related pathways, including pathways relevant to NF‐κ B signaling, IL‐17 signaling, MAPK signaling, RAS signaling, and Toll‐like receptor signaling, were activated by both MTB and MB attacks. Importantly, only RAP1, MAPK, RAS, and PI3K‐Akt signaling pathways were activated by MB attacks, which suggested that MB could evade the host immune system by certain strategies. Signaling‐related pathways are known to be important parts of immune system of the host that are involved in defense against pathogen infection 31. Activation of MAPK signaling pathway is known to be inevitable for host response towards pathogens. In particular, it is important for the activation of immunomodulatory molecules, such as TNF‐α and IL, which have been previously shown to be important for macrophages to defend/fight MTB invasion 25. The present study also reported activation of NF‐κB, IL‐17, and Toll‐like receptor signaling pathways in MTB‐attacked macrophages. These pathways are known to be closely associated with host defense responses, such as various pro‐inflammatory responses and release of antimicrobial effectors 32. Additionally, activation of IL‐17 signaling pathway has been identified in MTB‐infected macrophages, and IL‐17 could regulate pro‐inflammatory response and chemokines 10,33. Moreover, activation of peripheral inflammatory cells could produce microbiocidal substances, such as TNF‐α, to inhibit pathogenic bacteria and protect the host cells 13. Besides this, it has been previously reported that MTB‐induced IFN‐γ, IL‐6, IL‐12, IL‐23/IL17, and other cytokines that performed important functions in response towards MTB, and promoted inflammation 10. The activation of chemokines could further accelerate the migration of different cell subsets to MTB infected tissues and promote granulomatous formation to kill MTB 34,35. The present study identified a bunch of MTB‐induced chemokines in macrophages, which included CCL2, CCL3, CCL5, CCL7, CCL12, CXCL2, CXCL8, and CXCL10. These chemokines were involved in pro‐inflammatory response and inhibited MTB. Importantly, protein interaction network showed that CXCL2 and CXCL3 could regulate lipid metabolisms by directly interacting with lipid‐related genes that were associated with MTB infection. Therefore, the study highlighted the role/involvement of chemokines in the interaction mechanisms of MTB and macrophages.
Altogether, the presented integration analysis of transcriptome and lipid metabolomics on BAM and MTB/MB revealed the involvement of different mechanisms in the interaction between BAM and MTB/MB. The study proved that disorder of lipid metabolism in BAM, which resulted in the FM formation, was associated with the pathogenesis of MTB and MB. Importantly, BAM could deploy a series of signaling pathways and chemokines to defend/fight against MTB infection, whereas MB could evade only a part of BAM defensive responses. These results provided deeper insights into different defense responses of BAM towards MTB or MB infections, which would further assist in the development of novel strategies for the treatment and detection of tuberculosis in the future.