Interleukin-10 Family and Tuberculosis: An Old Story Renewed.

The interleukin-10 (IL-10) family of cytokines consists of six immune mediators, namely IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26. IL-10, IL-22, IL-24 and IL-26 are critical for the regulation of host defense against Mycobacterium tuberculosis infections. Specifically, IL-10 and IL-26 can suppress the antimycobacterial immunity and promote the survival of pathogen, while IL-22 and IL-24 can generate protective responses and inhibit the intracellular growth of pathogen. Knowledge about the new players in tuberculosis immunology, namely IL-10 family, can inform novel immunity-based countermeasures and host directed therapies against tuberculosis.

Introduction

Tuberculosis(TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of morbidity and mortality globally 1. The chronicity of tuberculosis mirrors the complex interaction between the pathogen and the host 2. The HIV coinfection and emergency of multidrug and extensive drug resistant Mtb further exacerbated the predicament of tuberculosis control 3. Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the only vaccine available for TB, was plagued by its limited and sometime unstable efficacy 4.

Cytokines are critical for immunity against foreign invaders 5. The interleukin (IL)-10 family of cytokines includes six immune mediators (IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26) with similar protein motif, receptor complex and conserved signaling cascades 6, 7. Their biological effect is diverse and widely ranges from anti-inflammatory to pro-inflammatory 6. Two subfamilies exist based on the biological effect, immune regulatory cytokines including IL-10 only, and IL-20 subfamily consisting of the remaining five members (IL-19, IL-20, IL-22, IL-24 and IL-26) 8. The genes encoding IL-10, IL-19, IL-20 and IL-24 are located on the chromosome 1q32 6. Whereas, IL-22 and IL-26 encoding genes cluster with IFN-gamma gene on the chromosome 12q15 6. IL-10 signals via IL-10RA/IL-10RB 9 and results in the phosphorylation of Stat3, a critical transcription factor for the immunosuppressive effect of IL-10 10. IL-19, IL-20 and IL-24 induced their signaling through the IL-20RA/IL-20RB receptor complex. However, IL-20 and IL-24 preferentially engage with the IL22RA1/IL-20RB receptor complex. IL-22 and IL-26 function via receptor complex consisting of IL-22RA1/IL-10RB or IL-20RA/IL-10RB 11. Interestingly, IL-20 subfamily can also activate Stat3, but the outcome is different from the activation of IL-10 11. The molecular mechanisms underlying the diverse effect of Stat3 signaling remain poorly understood.

The role of IL-10 family of cytokines in tuberculosis has been addressed recently 12-16. Here we discussed the roles of IL-10, IL-22, IL-24 and IL-26 in the context of tuberculosis.

Cellular source and mycobacterial regulation of IL-10 family of cytokine

IL-10 can be produced by hematopoietic and non-hematopoietic cells in response to Mycobacterium tuberculosis Complex infection (Figure 1). BCG can activate the pattern recognition receptor (PRR) on the surface of alveolar epithelial cells. The engagement of TLR2 and TLR4 on surface of epithelial cells by BCG can activate the glycogen synthase kinase-3 (GSK3) by PI3K-Akt and promote the production of IL-10 and IL-22 17. However, conflicting evidence is that the suppression of GSK3 by chemical inhibitor can significantly boost the BCG induction of IL-10 derived from macrophages 18.

Figure 1

Mycobacterium tuberculosis or M.bovis BCG can engage with PRR on the hematopoietic and non-hematopietic cells and result in the activation of downstream effectors and IL-10 expression. Receptors and signaling involved in the expression of IL-20 subfamily in response to Mtb infection is currently unknown. → Direct activation; ↵ = autocrine; ? = unknown

In neutrophils, coactivation of TLR2-MyD88- and C-type lectin receptor (CLR)- spleen tyrosine kinase (Syk)-dependent pathways led to the phosphorylation of p38 MAP and Akt kinases and subsequently, the upregulation of IL-10 19.

The interaction between the BCG-infected dendritic cells (DCs) with neutrophil through CD11b can activate MyD88 and Syk, to produce large quantities of IL-10 20.

Mtb can upregulate the IL-10 production via TLR2- ERK pathway 21. Activation of ERK by Mtb Rv1265 was critical for induced IL-10 expression in macrophages 22. Binding of the Mtb heat shock protein 60 to the TLR2 can activate macrophage p38 MAPK to increase the IL-10 production 23. Similarly, the engagement of Mtb PPE32 with TLR2 can upregulate the IL-10 production via co-activation of NF-κB and MAPK 24. Taken together, TLR2 and its downstream signaling MAPK pathway are essential for the induction of IL-10 expression in myeloid cells in response to the M. tuberculosis infection 25.

TLR3 regulates IL-10 expression in response to Mycobacterial-RNA through the PI3K/AKT signaling pathway 26. BCG-stimulated IL-10 expression in human macrophages by activation of dsRNA-activated serine/threonine protein kinase (PKR), which in turn activates MAPK and NF-κβ 27.

Mtb promoted chemokine receptor 5 (CCR5) expression in macrophages, which in turn increased IL-10 production 28.

It was found that IFN-α can induce IL-10 production by macrophages through the phosphorylation of Stat1 29. IFN-α/β can induce IL-10 production by Mtb-infected macrophages 30. This was supported by a murine study demonstrating that suppression of IFN-α/β by TPL-2-ERK1/2 signaling led to inhibition of IL-10 production 31.

Mtb infected antigen-presenting cells (APCs) can activate DAP12 (DNAX-activating protein of 12kDa) required for IRAK-M (interleukin-1 receptor-associated kinase M) expression, which in turn induces IL-10 production 32. BCG can enhance the production of IL-10 by binding to the DC-SIGN in DCs 33. Similarly, Mannose-capped lipoarabinomannan (Man-LAM) can induce IL-10 production through targeting the Dectin-2 in APCs 34. BCG induced high levels of IL-10 production by cord blood DCs via upregulation of the nuclear transcription factor Rel-B 35.

Mtb can induce IL-10 production by CD8+ T cells 36, 37 {Cyktor, 2013 #1}{Cyktor, 2013 #1}. CD4+ T cells produced high levels of IL-10 in response to purified protein derivative (PPD) 38.

IL-22 is produced by cells of the lymphoid lineages in response to Mycobacteria, which includes CD4+ cells (Th17 and Th22) 39, 40 and natural killer (NK) cells 41 (Figure 1).

Stimulation of NK cells with Mtb in the presence of IL-15 led to DAP10 expression and subsequently, IL-10 production 42. Mtb PstS1 upregulated IL-22 secretion from memory CD4+ T was mediated via DCs 43.

However, regulatory B cells can suppress IL-22 production 44. The production of IL-22 showed significant increase in the absence of IFN-γ 45.

IL-24 is mainly produced by monocytes and activated T cells 46, 47(Figure 1).

IL-26 is produced by myeloid and lymphoid lineages of white blood cells in response to Mtb infection 13(Figure 1). IL-26 can be released by Th17 48, 49, Th1 cells 48 and NK cells 50. However, how Mtb or its antigens regulate the expression of IL‑22, IL-24 and IL-26 remains to be elucidated.

Role of IL-10 family in Mycobacterial immune evasion

IL-10 is a potent immunoregulatory cytokine indispensable for the host immunity against mycobacteria infection (Figure 2). Consistently, IL-10 transgenic mice fail to control Mtb infection and develop severe lung pathology compared with nontransgenic littermates 51. However, IL-10-knockout mice were more resistant to Mtb infection and can limit bacterial growth within lung and spleen in comparison with the wild type 52. Blocking of murine IL-10 signaling during Mtb infection was beneficial for the control of the growth of bacteria and enhanced the survival of mice 53. It was shown that IL-10 -/- mice exhibited stronger Th1 responses during Mtb infection with concomitant massive pulmonary inflammation 54. However, conflicting data is reported on the immunomodulatory effect of IL-10 during Mycobacterial infection. IL-10 transgenic mice show normal Th1 response to Mtb infection, but IL-10 can reduce the production of antimicrobials reactive nitrogen intermediates (NO) by macrophages via increased arginase-1 (arg1) expression. This led to the progression of the disease and earlier death of mice 55. Furthermore, the expression of arg1 in hypoxic granuloma regions led to decreased T cells expansion 56.

Figure 2

Regulation of immune responses by IL-10 family cytokines during Mycobacteria infection: IL-10 family of cytokines induced by mycobacteria infection modulates immune responses. IL-10 can enhance intracellular survival and growth of bacilli by inhibition nitric oxide production, phagosomal maturation and IFN-γ-mediating killing. In addition, IL-10 can inhibit CD4+ T cell responses by suppression of IL-12 production and trafficking of Mtb peptide-MHC-II complexes to the plasma membrane. It also suppresses T cells cross-priming by antigen-presenting cells via blocking macrophages apoptosis. IL-10 can modulate granuloma formation via inhibition of TNF-α, Th17 cells and T cells expansion. Il-26 enhances intracellular survival of Mtb by unknown mechanism. IL-26 can recruit neutrophils to site of battle that can cause tissue damage and suppression Th1 response. IL-26 also promotes binding of released bacterial DNA to the TLR9 in surface of plasmacytoid DC resulting in production type I IFN. IL-22 can inhibit intracellular growth of bacilli via enhancing phagolysosomal fusion. IL-22 is an important activator of adaptive immune responses through promoting the expansion of memory CD4+ cells and IFN-γ production and recruitment of B cells to site of infection. IL-24 induces IFN-γ production by CD8+ cells and suppresses FOXP3 T reg cells.

The absence of IL-10 enhanced the production of IL-17, and resulted in excessive lung inflammatory response 12. Similarly, blockade of IL-10 during BCG vaccination can increase the protection against Mtb challenge in both susceptible and comparatively resistant strains of mice due to enhanced Th17 cell responses 57. IL-10 can repress 17A production through direct interacting with IL-10Rαin Th17 cell 20. IL-17A deficient mice failed to develop mature granuloma and protective responses against virulent Mtb infection 58. However, IL-10-deficient mice infected with Mtb can generate mature fibrotic pulmonary granulomas and improve the control of bacterial growth in the lungs 16. Furthermore, IL-10 suppresses TNF-α production 30, 59, 60, TNF-αis essential for granulomas formation and blocking of TNF-αsignaling leads to the disintegration of granuloma and dissemination of Mtb 61. Moreover, IL-10 can suppress the formation of multi-nucleated giant cells (MGC) from monocytes of TB patients in response to PPD 62, the histologic hallmark of granuloma which can limit Mtb infection. Taken together, the data suggest that IL-10 promotes the persistence and survival of pathogens. Therefore, the modulation of IL-10 levels might facilitate the TB elimination.

Peripheral blood mononuclear cells (PBMCs) obtained from multi-drug resistant TB patients stimulated by ESAT6 displayed a lower frequency of IFN-γ-producing T cells and a higher frequency of regulatory T cells (Treg) correlated with increased IL-10 secretion, when compared with cells from non-tuberculous Mycobacteria patients. Blockage of IL-10 or IL-10Rα with antibody can enhance Th1 responses and decreases the Treg cells 63.

Macrophages apoptosis is important for the cross-priming of T cells by DCs. DCs can uptake the apoptotic vesicle from the infected apoptotic macrophages and subsequently prime the naïve T cells 64. IL-10 can suppress Mtb-induced macrophages apoptosis by reducing TFN-αand upregulating of Bcl3, an inhibitor of NF-κB activity 60. Similarly, inhibition of macrophages apoptosis by virulent M. bovis was shown to be correlated with increased IL-10 and Bcl2 production and decreased TNF-α production 65. Taken together, Mycobacteria can subvert the apoptosis of macrophages for better survival and persistence.

IL-10 can inhibit the production of IL-12 in Mtb-infected macrophages 30. IL-10 can interfere with antigen presentation by inducing MARCH1 member of ubiquitin ligase family that interacts and degrades class II major histocompatibility complex (MHC-II) molecules 66. It has been shown that IL-10 pretreated monocyte-derived macrophages (MDMs) failed to traffic the Mtb peptide-MHC-II complexes to the plasma membrane 67. IL-10 also inhibits MHC-II expression in Mtb-infected macrophages via activation of Stat3 28. Suppression of IL-12 and MHC-II by IL-10 in Mtb-infected macrophages led to compromised the Th1 responses 21. Consistently, blocking of IL-10 signaling in Mtb-infected macrophages can enhance IL-12 secretion and augment the IFN-γ production by CD4+ T cells 68.

IL-10 also can enhance Mtb survival and growth in macrophages by suppressing phagosomal maturation partially dependent on Stat3 activation 69. Taken together, IL-10 enhances mycobacterial intracellular survival and growth by suppressing innate and adaptive immune response.

The role of IL-26 in host defense against Mtb has been explored very recently (Figure 2). The possible role of IL-26 in Mtb infection has been demonstrated by ex vivo study 13. Levels of IL-26 are lower in the Mtb-infected monocytes supernatants from TB patients than from healthy control 13. The addition of recombinant IL-26 to whole blood infected with Mtb led to enhanced bacterial survival and growth 13. However, how Mtb downregulates IL-26 and its role in antimycobacterial response remain to be explored.

IL-26 has quite unexpected cationic antimicrobials peptides properties that can lyse extracellular bacteria by forming membrane-pore 70. Also, it can bridge the released bacterial DNA and TLR9, thereby triggering the production of type I IFN by plasmacytoid DCs 70. IFN-α/β are potent inhibitors of the IFN-γ responsiveness and the production of type I cytokines that are required for protection against Mtb infection 48.

IL-26 produced by alveolar macrophages can recruit the neutrophils via induction of chemotaxis factors such IL-8 and leukotaxin n-formyl-methionyl-leucyl-phenylalanine (fMLP) 71. IL-26 also can inhibit random migration (chemokinesis) of neutrophils 71. Depletion of the neutrophil at the onset of TB infection results in significant increase the numbers of IFN-γ-producing CD4 T-cells, correlated with reduced bacterial load and lung pathology 72. In-depth understanding of IL-26 biology is crucial to the development of immune intervention against tuberculosis.

Role of IL-10 family in immune protective against Mycobacteria infection

It was predicted that the production of low levels of IL-10 by activated macrophages is required to an efficient antimicrobial response, control of bacterial growth and to prevent lung damage 73. Therefore, modulation of IL-10 levels during TB therapy might shorten the duration of the treatment and accelerate the bacterial clearance.

IL-22 is crucial for tissues modulation in responses to inflammation. It induces proliferative and anti-apoptotic pathways, as well as anti-microbial substances, that can enhance tissue repair and prevent its damage 11.

IL-22 has been shown to be significantly upregulated in the blood from juvenile rhesus monkeys with severe TB disease 74. IL-22-producing T cells were found most frequent localized in lungs than in blood and lymphoid tissue, and involved in granuloma formation in macaque TB model 15. Interestingly, IL-22 was found to be most significantly upregulated in PBMCs from vaccinated-protected cattle, when compared with vaccinated-unprotected and unvaccinated control, suggested that IL-22 a potential biomarker for vaccine success 75. IL-22-deficient mice display normal response to Mtb infection 76. However, IL-22 may trigger the recruitment of B cells to the lungs by upregulating CXCL13 expression 77. B cells play crucial roles in host defense by reducing inflammation and containment of Mtb 78. IL-22-knockout mice challenged with Mtb exhibited smaller B cell follicles in comparison with the counterpart 77.

Administration of IL-22 into the mice at the time of BCG vaccination led to reduced bacterial burden and enhanced CD4+ T cell responses after Mtb challenge 41.

A polymorphism (rs2227473) in the IL-22 promoter was shown to be associated with TB susceptibility in Chinese populations 79. IL-22 producing CD4+ T cells (Th22 ) frequencies were lowered in PBMCs from TB pleurisy (PTB) patients in comparison with healthy, whereas, IL-22 was detected in high concentration in bronchoalveolar lavage (BAL) fluid obtained from TB patients 80. Likewise, the Th22 cells are elevated in tuberculous pleural fluid 40, 81, suggested that IL-22 does involve in immunity at local site of Mtb infection. In HIV patients with TB disease, IL-22 was differentially induced in PBMCs collected from TB with reconstitution inflammatory syndrome (TB-IRIS) after in vitro stimulation with Mtb, and higher concentration of IL-22 proteins was detected in their serum, when compared with non-IRIS patients 82. Taken together, these data suggest that IL-22 might play a decisive role in tuberculosis immunity.

Modern Beijing strains can stimulate PBMCs from healthy individuals to produce lower level of IL-22 than the ancient Beijing and Euro-American strains 83. Manipulation of IL-22 level by Mtb modern Beijing strains might benefit its intracellular survival.

IL-22 plays indispensable role in innate and adaptive immune defense against Mtb infection (Figure 2), production of IL-22 by NK cells can inhibit intracellular growth of Mtb through enhancing phagolysosomal fusion 42. Interesting, IL-22 can be found in the membrane of mature CD4+ T effector cells and the direct interaction of these cells with Mtb-infected macrophages led to suppress intracellular bacterial growth 84. However, the molecular mechanisms by which IL-22 inhibits intracellular growth of Mtb are to be determined. Recently, it was found that IL-22 induced calgranulin A expression that can enhance phagolysosomal fusion by increasing Rab7 expression and downregulatiing Rab14 expression 85.

IL-24, also known as melanoma differentiation associated gene 7 product(mda-7), has potent antitumor activity through selectively induced tumor death without detriment effect in normal cells 86. Levels of IL-24 are significantly lower in the serum of TB patients in comparison with healthy volunteers 14. IL-24 expression was decreased in PBMCs infected with Mtb, and addition of recombinant IL-24 can induce IFN-γproduction by CD8+ T cells 14.

IL-24 was significantly upregulated in PBMCs obtained from BCG-Japan strain-vaccinated children stimulated with culture filtrate proteins (CFP) of Mtb compared with children vaccinated with BCG-Brazil or BCG-Denmark strain 87. PBMCs obtained from latent TB patients (LTB) produces more IL-24 than from active TB patients 88. Furthermore, exogenous IL-24 can increase IFN-γin PBMCs obtained from TB patients, however, neutralization of IL-24 can reduce IFN-γexpression in PBMCs collected from LTB patients 46. IL-24 increased IFN-γexpression through increasing expressions of IL-12, 1L-23 and IL-27, and by decreasing FOXP3 expression 46 (Figure 2). Taken together, Mtb suppresses IL-24 expression might enhance its persistence host tissue. However, underlying molecular mechanisms remain to be explored.

Conclusions

In conclusion, IL-10 family of cytokines has diverse roles during Mycobacteria infection ranging from immune evasion to immune protective. The absence of IL-10 can enhance immune response and early clearance of Mtb. IL-10 can enhance the intracellular survival of bacilli via several means, namely 1) inhibiting phagosomal maturation, 2) reducing nitric oxide production and 3) blocking IFN-γsignaling in macrophage. In addition, IL-10 can antagonize the development of Th1 responses through suppressing antigen presentation and IL-12 production. Moreover, it can inhibit macrophages apoptosis that is require for efficient development of T cells responses. However, low level IL-10 can trigger antimicrobial response and prevent tissue damage. Therefore, decreasing the IL-10 level during TB therapy might benefit for the rapid clearance of bacteria.

IL-26 is a novel player with scarce information in TB immune responses; it can limit Mtb clearance during in vitro infection of PBMCs by mechanisms currently unknown. Understanding how IL-26 enhances intracellular growth of bacilli is critical to develop better countermeasures against this notorious pathogen.

IL-22 plays a decisive role in innate and adaptive immune responses against Mtb infection by promoting phagolysosomal fusion, expansion and IFN-γproduction by memory CD4+ cells and recruitment of B cells.

IL-24 induces IFN-γproduction by CD8+ T cells. IL-22 and IL-24, crucial for the coordinated cell-mediated immunity, might represent good candidates for better TB therapy.