The Biology and Role of Interleukin-32 in Tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality globally, with nearly 10.4 million new cases of incidence and over 1.7 million deaths annually. Drug-resistant M. tuberculosis strains, especially multidrug-resistant or extensively drug-resistant strains, have further intensified the problem associated with tuberculosis control. Host-directed therapy is a promising alternative for tuberculosis control. IL-32 is increasingly recognized as an important host molecule against tuberculosis. In this review, we highlight the proinflammatory properties of IL-32 and the mode of action of IL-32 in mycobacterial infections to inspire the development of novel immunity-based countermeasures and host-directed therapies against tuberculosis.

The secretion of IL-32 isoforms remains to be investigated. IL-32γ possesses an N-terminal hydrophobic signal peptide, which is a typical feature of secreted cytokines. IL-32 is expressed in peripheral blood mononuclear cells (PBMCs) by LPS stimulation or M. tuberculosis infection, instead of Staphylococcus aureus and Candida albicans [58]. The IL-32α isoform was detected as an intracellular fraction, whereas the IL-32β isoform was found in the cell culture supernatant of Cos7 cells under transient transfection [3]. However, when performing transient transfection of IL-32β into bovine aortic vascular endothelial cells (BAVECs), IL-32β was found mainly in the cytosol and localized in the endoplasmic reticulum [6]. In addition, IL-32β was detected in the supernatant derived from the cytoplasm of apoptotic T cells but not secreted in anti-CD3 antibody-activated human T cells [12]. However, IL-32 can bind to the RGD motif of integrin, and IL-32 isoforms contain predicted tyrosine sulfation sites, which are prevalent in secreted proteins [2,5,59]. In HT-29 cells stimulated with TNF-α and IFN-γ, IL-32 was associated with membrane vesicles, and the release of IL-32 depended on exosomelike vesicle release mechanisms [60]. Therefore, IL-32 may be secreted via a nonclassical protein secretion pathway, similar to IL-33 and HMGB1, without typical signal peptides and are released via ER/Golgi-independent means [60,61].
Four major isoforms (IL-32α, IL-32β, IL-32γ, and IL-32δ) were found in IL-2-stimulated human NK cells [3]. IL-32β, IL-32ε, and IL-32ζ were isolated from activated T cells [12], and IL-32s expression was first observed in Jurkat human leukemia T cells [70]. IL-32ε, IL-32ζ, IL-32θ, and IL-32s are also found in T cells, and the IL-32β isoform is mainly expressed in activated T cells [2,12,46]. IL-32θ and IL-32s were identified from monocyte-derived dendritic cells purified from human PBMCs and Jurkat T cells via 5 ′ RACE [46]. The function of different IL-32 isoforms in different cell types was summarized in Table 1. IL-32 mRNA levels increased after stimulation with Con A and monoclonal antibodies against CD3 and CD28 [62]. TNF-α reciprocally induced the expression of IL-32 mRNA in monocytederived dendritic cells, T cells, and synovial fibroblasts [62]. Intracellular IL-32 is constitutively expressed in human umbilical vein endothelial cells (HUVECs). The IL-32α and IL-32γ isoforms are the most prominently expressed IL-32 mRNAs in unstimulated endothelial cells [6,60,68,77], while TNF-α and IL-1β induced the expression of IL-32β in endothelial cells [4]. Studies have shown that GM-CSF induces the expression of the IL-32α, IL-32β, IL-32γ, and IL-32δ isoforms in a caspase-1-dependent manner in eosinophils [15,16]. Synovial fibroblasts isolated from patients with rheumatoid arthritis express IL-32γ after stimulation with IL-1β and TNF-α [48]. TNF-α can also promote the expression of the IL-32α, IL-32β, IL-32δ, and IL-32γ isoforms by activating the Syk/PKCδ/JNK/c-Jun signaling pathway [69]. The cell or tissue-specific expression patterns and functions of each isoform of IL-32 remain to be determined.

IL-32 Regulates the Expression of MicroRNAs
IL-32 isoforms were shown to induce inflammation by regulating the expression of microRNAs [20,37,92,93]. The expression of IL-32 is activated by human cytomegalovirus infection and functionally downregulated by hcmv-miR-UL112-1 [37]. MiR-23b-3p directly targets and induces the expression of PTEN, resulting in reduction in PI3-kinase, total Akt, and IL-32 levels [93]. IL-32α promotes the expression of the atheroprotective-associated genes Timp3 and Reck by downregulating the Rprd2-Dgcr8/Ddx5-Dicer1 biogenesis axis downstream of microRNA-205 [92]. Overexpression of human IL-32γ in transgenic mice led to increased bone formation, reduced bone loss with advancing age, and high osteogenic capacity of osteoblasts by upregulation of microRNA-29α [20]. Therefore, IL-32 is a novel protective cytokine that acts against mycobacterial infection. Elucidating the complex interactions between the IL-32 isoforms, microRNA-based regulation of the isoforms and the function of IL-32 will provide novel insight into the novel mechanism of the protective roles of IL-32 in multiple diseases.

The Function of IL-32 in Mycobacterial Infection
M. tuberculosis, the causative agent of human TB, can subvert host immune defenses to promote its own intracellular survival. Infection of human macrophages or PBMCs with M. tuberculosis H37Rv induced IL-32 production [11,58], suggesting a role for IL-32 in the control of M. tuberculosis infection. M. tuberculosis and Mycobacterium bovis induced the release of IL-32 from PBMCs via IFN-γ, which was produced after caspase-1-activated IL-18 release [58]. Silencing of endogenous IL-32 in differentiated THP-1 human macrophages significantly decreased TNF-α, IL-1β, and IL-8 production and simultaneously increased the M. tuberculosis burden in infected macrophages [11]. The antimycobacterial effect of IL-32 may be partly due to enhanced cell apoptosis in infected macrophages. IL-32γ is a potent inducer of apoptosis; both IL-32γ and IL-32β can induce caspase-3-and caspase-8-dependent apoptosis [12,27]. Endogenous IL-32 mediated M. tuberculosisinduced apoptosis of macrophages, suggesting that apoptosis of infected macrophages is a mechanism to protect against mycobacterial infection. IL-32γ decreased the M. tuberculosis burden within macrophages via classic caspase-3-mediated apoptosis [11] and caspase-1-or lysosomal-cathepsinmediated apoptosis [94]. Our previous study showed that M. tuberculosis PE/PPE (Pro(P)-Glu(E) and Pro(P)-Pro(P)-Glu(E)) family antigen PPE32 induced ER-stress-mediated cell apoptosis via the stimulation of IL-32 production [95]. In addition, IL-32 serves as a mediator of IFNγ-vitamin D-related antimicrobial activity and a marker for latent TB infection (LTBI), as determined via the mining of TB transcriptomic datasets [96]. IL-32γ was also found to be associated with the vitamin D antimicrobial pathway in human macrophages [84]. IFN-γ-induced IL-32γ increases the expression of the vitamin D receptor, leading to the expression of cathelicidin and β-defensin 2 (DEFB4), which are potent antimicrobial peptides that act against intracellular infection in macrophages [84]. IFN-γ treatment activates the production of NO in macrophages, which is the main microbicidal molecule involved in the control of M. tuberculosis infection [97]. Human THP-1 cells express iNOS and produce NO after differentiation into macrophages by treatment with IL-32γ [98]. The production of reactive oxygen species (ROS) is required to induce the microbicidal activity mediated by vitamin D and cathelicidin, and cathelicidin enhances the production of ROS and proinflammatory cytokines, such as TNF-α, IL-8, and IL-6 [99]. M. tuberculosis-induced GM-CSF can promote NO production and phagolysosomal fusion against M. tuberculosis infection [100,101]. GM-CSF might kill intracellular M. tuberculosis via induction of IL-32 as GM-CSF increases the expression of IL-32 in other cell types [15,16]. In summary, IL-32γ is a protective molecule that enhances the microbicidal activity of macrophages against M. tuberculosis via increased apoptosis and pyroptosis, and antimicrobial peptides induced by vitamin D and GM-CSF are involved in protection against M. tuberculosis infection (Figure 1). IL-32, lacking sequence homology with known cytokine families, is a novel proinflammatory cytokine [3]. The expression of IL-32 was increased in patients with M. avium infection [42]. IL-32γ significantly reduced the intracellular survival of M. avium in human monocyte-derived macrophages [42]. Moreover, the expression of endogenous IL-32 and NOD2 was increased in patients with the restrictive tuberculoid form of leprosy, which is caused by Mycobacterium leprae infection [102], suggesting that both NOD2 and IL-32 are associated with leprosy. IL-32 expression was increased in surgically resected lungs of active TB patients, particularly in airway epithelial cells and granuloma macrophages [43], suggesting a protective role of IL-32 against in vivo M. tuberculosis infection. However, there was a decrease in the protective response of IL-32γ against M. tuberculosis at later time points of infection as IL-32γ mRNA is spliced into IL-32β, leading to increased levels of IL-10-expressing macrophages or DCs in the lungs [43].

Conflicts of Interest
The authors declare that they have no competing interests.