Lung epithelial cells interact with immune cells and bacteria to shape the microenvironment in tuberculosis

Epithelial sensing of Mtb

EC express a large repertoire of pattern recognition receptors (PRRs) to sense micro-organisms. These include the membrane-bound Toll-like receptors (TLRs) and C-type lectins, cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors and nucleic acid sensors (table 1). On contact with pathogens, PRRs are activated and EC secrete an array of pro-inflammatory and anti-inflammatory cytokines as well as chemokines which attract different immune cell subsets (table 1). Interestingly, CD4⁺ T cell-associated type 1 helper (Th1) and Th2 cytokines were also shown to regulate expression of different TLRs in primary airway EC (PBEC), illustrating the complex cytokine milieu required to coordinate appropriate responses to pathogens.²¹ In addition, antimicrobial peptides (AMPs) with direct effects on pathogens are secreted by airway and alveolar epithelium (table 1). For example, β-defensins are AMPs with broad-spectrum activity against bacteria, fungi and certain viruses and are produced by airway and alveolar EC in response to mycobacterial infections. Human β-defensin-2 (hBD2) controls Mtb growth²² and has chemotactic activity for memory lymphocytes and immature dendritic cells (DCs).²³ LL-37, a cathelicidin AMP, is also produced by PBEC, AM and the AEC2-like cell line A549 (an overview of epithelial and immune cell lines that are used in culture models is provided in online supplemental table S1) in response to mycobacterial infection.^{24 25} LL-37 and β-defensin-2 (hBD2) expression can be upregulated by vitamin D, and physiological vitamin D levels contributed to restriction of Mtb or Mtb/HIV replication in macrophages.²⁶ Interestingly, vitamin D-induced interleukin-1 (IL)-1β secretion by macrophages stimulated antimycobacterial capacity of primary small airway EC in a coculture model and reduced bacterial burden.²⁷ These effects were dependent on epithelial production of hBD2 and IL-1 receptor type 1 signalling. On the other hand, IL-1-mediated crosstalk between EC and Mtb-infected AM was shown to promote dissemination of infected cells.²⁸ Other products secreted by EC might also have indirect effects on mycobacteria. For example, various hydrolases secreted in the alveolar surfactant were shown to modify the Mtb cell wall.²⁹ This resulted in a significant decrease in Mtb association with and infection of macrophages.

Different epithelial cell types employ various defence strategies that contribute to clearing infections. Secretory goblet cells produce mucus which is mechanically transported up the airways by ciliary motion of multiciliated cells,³⁰ resulting in physical removal of the pathogen. AEC2 secrete surfactant containing the aforementioned antimicrobial mediators and reactive oxygen species, to keep pathogens, including Mtb,³¹ at bay. EC can therefore be considered sentinels, initiators and modulators of immunity, thus contributing directly to infection-driven pathogenesis, including the regulation of innate and adaptive immune responses.

Epithelial infection by Mtb

Besides sensing pathogens and acting as an early warning and defence system, EC of the lung can also be directly infected by mycobacteria. PBEC seemed relatively insensitive to infection with Mtb³² in vitro, though Mycobacterium avium and Mycobacterium abscessus achieved higher infection rates when used in supraphysiological quantities.³³

In contrast to the bronchial EC described above, alveolar EC might be more susceptible to Mtb infection. Early reports described successful infection of the cell line A549 by Mtb and the attenuated vaccine strain Mycobacterium bovis Bacille Calmette-Guerin (BCG).^{34 35} Moreover, both species were able to migrate across a bilayer model of A549 and endothelial cells, in the absence of macrophages.^36–38 During migration of Mtb, bacteria were observed intracellularly.³⁷ Cell death and a decreased capacity to maintain cellular tight junctions was indicated by a drop in transmembrane electrical resistance (TEER) and increased passage of dextran through the cell layer.^{36 37} Therefore, it appears that Mtb kills infected cells to gain passage through the alveolar barrier. Another study using primary rat alveolar cell layers found a marked decrease in TEER on Mtb infection, which appeared to result from excessive tumor necrosis factor - alpha (TNF-α) production by the epithelium.³⁹ Furthermore, dissemination-attenuated strains with decreased migratory capacities demonstrated a role for Mtb virulence factor ESAT-6 in the direct crossing of the alveolar barrier.³⁸ These studies only included A549 cells and validation in primary alveolar EC will be important.

EC are non-professional phagocytes, although mechanisms of mycobacterial adherence and internalisation by EC have been described. The proteoglycan syndecan 4 (SDC4) was identified as an attachment receptor for BCG on alveolar A549 cells, and this interaction involved host SDC and mycobacterial heparin-binding haemagglutinin adhesin.⁴⁰ Furthermore, SDC4-knockout mice were more resistant to Mtb H37Rv colonisation.⁴⁰ Other adhesins expressed by Mtb that directly bind EC are malate synthase and ESAT-6, which may also facilitate dissemination of mycobacteria by epithelial barrier disruption via cell lysis.³⁸

Inside EC, Mtb is initially processed via the phagocytic pathway, where it is localised in late endosomal compartments in alveolar and airway epithelium.^{41 42} However, fusion of Mtb-containing compartments with lysosomes was blocked in EC, as is the case in macrophages. In A549 cells, Mtb was found inside vesicles labelled with the autophagy marker LC3, indicating that Mtb is processed via the autophagy pathway.⁴¹

Innate immune cell activation by Mtb-exposed epithelium

Sometimes the direct response mounted by the lung epithelium is not sufficient to eradicate invading pathogens. EC also secrete inflammatory mediators that may affect the type and magnitude of the immune response that follows. Culture of the monocyte cell line U937 in presence of Mtb-infected A549 cells resulted in reduced Mtb-triggered TLR signalling in the monocytes, compared with infected monocytes alone.⁴³ This suggests that EC influence the inflammatory response of immune cells. Additionally, submerged PBEC cultures produced anti-inflammatory cytokines IL-10 and IL-22 in response to BCG infection.⁴⁴ This suggests that airway and alveolar EC act as moderators of inflammation during mycobacterial infection. In line with these observations, some studies found increased levels of IL-10 in sputum of patients with TB. This correlated with higher Mtb antigen burden,⁴⁵ suggesting that IL-10 could undermine effective clearance of Mtb. This effect is likely caused by the negative influence of IL-10 on Th1 T cells and macrophages,⁴⁶ which play a pivotal role in protective immunity against Mtb.

Neutrophils are attracted more strongly to PBEC infected with Pseudomonas aeruginosa (common in cystic fibrosis patients) than with a non-pathogenic Escherichia coli strain.⁴⁷ In this manner, EC filter pathogenic signals, levelling subsequent immune activation by moderating PRR expression.⁴⁸ A tightly regulated balance is important as EC recruiting too many leukocytes risk excessive inflammation and lung injury as a result. Neutrophils, for example, are considered protective during early Mtb infections, but pathogenic at later stages where they are associated with poor prognosis. CXCL5 is a chemokine produced by EC and important in the initial recruitment of neutrophils. Cxcl5 ^−/− mice exhibited enhanced survival after Mtb infection compared with wild-type mice. This resistance to Mtb infection was due to impaired neutrophil recruitment, which reduced pulmonary inflammation. This suggests that excessive epithelial-derived CXCL5 is critical for leukocyte-driven destructive inflammation in TB.⁴⁹

In turn, innate immune cells influence the rate of infection in EC. Mtb and BCG first cultured intracellularly in murine macrophages had a higher association with A549 cells and infected a higher percentage of them,³⁵ suggesting enhancement of mycobacterial virulence after exposure to the intracellular environment of macrophages. By contrast, Mtb and M. avium growth in the macrophage cell line Mono-Mac-6 was reduced by A549 cell-derived soluble factors including TNF-α and granulocyte-macrophage colony stimulating factor (GM-CSF).⁵⁰ Lastly, in a contact-independent model, PBEC were activated by Mtb-infected monocytes to produce antimicrobials and cytokines that were not inducible by direct infection of the bronchial cells themselves,³² highlighting the crosstalk between epithelial and immune cells.

Antigen presentation and T cell activation by the epithelium

Antigen presentation to T cells is required for activation of adaptive immune responses against Mtb. While this role is often fulfilled by DC, there is evidence for antigen presentation to T cells by EC. Several studies have identified expression of major histocompatibility complex class II (MHC-II) molecules on human bronchial (figure 3) and alveolar EC, which would imply direct activation of CD4⁺ T cells by the epithelium.^51–53 Human nasal and bronchial EC were able to stimulate allogenic T cells, an effect abrogated by addition of anti-MHC-II antibodies.⁵³ Functional MHC-II has also been identified on pulmonary EC of other species. Rats constitutively expressed MHC-II on AEC2, and to a lower degree on bronchial epithelium, suggesting cell type-specific regulation of expression.⁵⁴ Murine AEC2 also expressed MHC-II and were able to present antigens to primed CD4⁺ T cells when infected with Mtb.⁵⁵ Furthermore, expression of costimulatory molecules has been demonstrated in human epithelium and appears to be cell type specific. CD86 was found on both PBEC and A549 cells.^{52 56} CD80, on the other hand, was only found on alveolar EC.⁵⁶

CD8⁺ T cells play an additional role in protection against intracellular pathogens such as Mtb. In a study by Harriff et al, interferon gamma (IFN-γ) production by CD8⁺ T cells was stimulated by bronchial epithelium following Mtb infection.⁴² These authors also showed that the bronchial epithelial basal cell line BEAS-2B activated several Mtb-specific, classically (HLA-B45) and non-classically (HLA-E, MR-1) restricted T cell clones to a higher degree than DCs after infection with Mtb, as measured by IFN-γ producing T cells. This is perhaps a surprising observation, since bronchial epithelium was infected with lower efficiency compared with DCs.

Further studies highlighting the importance of EC in shaping T cell responses studied BCG vaccination in mouse and rhesus macaque models. In these studies, mucosal vaccination-induced superior protection against Mtb compared with intradermal vaccination.⁵⁷ Perdomo et al described the recruitment of significantly higher frequencies and absolute numbers of effector memory T cells (T_EM) and tissue-resident memory T cells (T_RM) after mucosal vaccination compared with subcutaneous vaccination.⁵⁸ Particularly the T_RM subset has been suggested to confer protection against infections, including Mtb.⁵⁹ T_RM cells are hardly detectable in the circulation, but abundantly present in tissues. Comparison of different administration routes showed substantially more antigen-responsive T_RM cells in all lung parenchymal tissues after intravenous BCG immunisation.⁶⁰ Furthermore, approximately 75% of all CD4⁺ and CD8⁺ T cells were T_RM following direct endobronchial instillation of BCG.

Other types of adaptive immune cells may participate in anti-Mtb immunity too. Mucosal-associated invariant T (MAIT) cells were found enriched in the airways,⁶¹ and Mtb-infected airway EC were capable of inducing IFN-γ production from MAIT cells.⁴² Furthermore, BCG vaccination induced transient expansion of the peripheral MAIT cell population in humans and specifically activated MAIT cells in non-human primates.^{62 63} Few studies to date looked at interactions between lung epithelium, regulatory T cells and innate lymphoid cells (ILCs). In Mtb-infected mice, IL-22-producing ILC3s prolonged survival and prevented epithelial cell damage.⁶⁴ Interestingly, EC may also regulate IL-17-producing T helper cells (Th17) via a complex feedback loop. The enzyme indoleamine-2,3-dioxygenase (IDO) is expressed by lung EC. Following IFN-γ release by T cells, downstream IDO products secreted by EC were found to selectively inhibit IL-17 production by Th17 cells.⁶⁵

Microfold cells (M cells) transport Mtb to bronchus-associated lymphoid tissue (BALT)

M cells are specialised EC present in areas of the airways where inducible BALT (iBALT) exists or can be induced.⁶⁶ These specialised M cells transport macromolecules and microbes from the airway into the subepithelial region, a process called antigen transcytosis. Subsequently, these antigens induce maturation of immature DCs, which in turn activate naive T cells residing in the iBALT. In mice, M cells enabled transport of particles and microbes, including Mtb, across the epithelial barrier.⁶⁷ Scavenger receptor B1 on M cells was reported to bind the major Mtb virulence factor EsxA, facilitating Mtb adherence to and translocation through the epithelial layer in vitro.⁶⁸ Furthermore, the TB granuloma is often surrounded by clusters of cells that are remarkably similar in structure to iBALT and perform tertiary lymphoid functions.⁶⁶ iBALT is not present in healthy adults and is formed following a period of inflammation in the airways. However, iBALT induced by a previous unrelated infection or allergic reaction, smoking or chronic inflammation (asthma) may persist for weeks or longer.^{66 69} Even so, it is unlikely that M cells and iBALT are present in healthy individuals when Mtb first enters their lungs, so the importance of M cells in initial Mtb infection biology is debatable. Although M cells have been induced in human bronchial epithelial cell lines⁶⁸ and their presence was demonstrated in multiple animal models, it has not been verified whether M cells or comparable cells do exist in human lungs. Taking these considerations into account, M cells could potentially function as gateways for Mtb (figure 3), though their relevance to human Mtb infection biology requires further investigation.

The findings discussed so far support the view of a tightly regulated interplay between the epithelium, immune cells and Mtb during different stages of infection. The next section of this review shall focus on possibilities to integrate these three key players in models for in vitro studies.