Complete ablation of tumor necrosis factor decreases the production of IgA, IgG, and IgM in experimental central nervous system tuberculosis

Document Type: Original Article


1 Program of Infection and Immunity, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhongshan School of Medicine, Sun Yat-sen University, Guangdong, China

2 Institute of Tuberculosis Control, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

3 Key Laboratory of Tropical Diseases Control, Ministry of Education, Sun Yat-sen University, Guangzhou, China

4 SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Stellenbosch University, Stellenbosch, South Africa

5 Division of Immunology, Department of Pathology and Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa

6 Institut de Transgenose, CNRS, GEM2358, Orleans, France, University of Orleans and CNRS UMR7355, Experimental and Molecular Immunology and Neurogenetics, Orleans, France

7 National Health Laboratory Service, Sandringham, Johannesburg, South Africa

8 Immunology of Infectious Disease Research Unit, South African Medical Research Council, Cape Town 8000, South Africa



Objective(s): This study aimed to explore the contribution of tumor necrosis factor (TNF) in the recruitment of B-cell and secretion of immunoglobulins (Igs) during cerebral tuberculosis (TB).
Materials and Methods: In this work, the contributing role of TNF in regulating Ig secretions was investigated by comparing wild type TNF (TNFf/f), B-cell-derived TNF (BTNF-/-), and complete TNF ablation (TNF-/-) in a mouse cerebral Mycobacterium tuberculosis infection. Using flow cytometry and ELISA, we were able to examine the recruitment of B-cell subsets, and the production of Igs; also assessed the expression of surface markers on B cell subsets.
Results: Here, we found that TNF-/- mice showed defective expression of IgA, IgG, and IgM antibodies compared with TNFf/f and BTNF-/- mice, which was significantly decreased in the expression of surface markers and co-stimulatory molecules. Moreover, mice that produced low antibody levels were not able to control infection, therefore progressed to disease; providing direct evidence for the TNF gene-regulating humoral immunity during central nervous system (CNS) M. tuberculosis infection. In contrast, BTNF-/- mice controlled the infection and had levels of IgA, IgG, and IgM comparable to TNFf/f mice.
Conclusion: Together, our results demonstrate that TNF may serve as an essential regulator of antibody-mediated immune responses in CNS TB. However, the protective level exhibited by TNF-producing B cells could be defined as baseline protection that could be used in the development of new therapeutic targets or designing new vaccines.


1. WHO, Global tuberculosis report 2018. World Health Organization 2018; 1-142.
2. Miotto P, Zhang Y, Cirillo DM, Yam, WC. Drug resistance mechanisms and drug susceptibility testing for tuberculosis. Respirology 2018; 1-16.
3. Be NA, Kim KS, Bishai WR, Jain SK. Pathogenesis of central nervous system tuberculosis. Curr Mol Med 2009; 9:94-99.
4. Garg RK. Tuberculosis of the central nervous system. Postgrad Med J 1999; 75:133-140.
5. Rock RB, Olin M, Baker CA, Molitor TW, Peterson PK. Central nervous system tuberculosis: pathogenesis and clinical aspects. Clin Microbiol Rev 2008; 21:243-261.
6. Misra UK, Kalita J, Nair PP. Role of aspirin in tuberculous meningitis: a randomized open label placebo controlled trial. J Neurol Sci 2010; 293:12-17.
7. Leonard JM, Des Prez RM. Tuberculous meningitis. Infect Dis Clin North Am 1990; 4:769-787.
8. O’Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in tuberculosis. Annu Rev Immunol 2013; 31:475-527.
9. Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol 2012; 12:352-366.
10. Volkman HE, Pozos TC, Zheng J, Davis JM, Rawls JF, Ramakrishnan L. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science 2010; 327:466-469.
11. Mardh PA, Larsson L, Hoiby N, Engbaek HC, Odham G. Tuberculostearic acid as a diagnostic marker in tuberculous meningitis. Lancet 1983; 1:367.
12. Mastroianni CM, Paoletti F, Lichtner M, D’Agostino C, Vullo V, Delia S. Cerebrospinal fluid cytokines in patients with tuberculous meningitis. Clin Immunol Immunopathol 1997; 84:171-176.
13. Tsenova L, Bergtold A, Freedman VH, Young RA, Kaplan G. Tumor necrosis factor alpha is a determinant of pathogenesis and disease progression in mycobacterial infection in the central nervous system. Proc Natl Acad Sci USA 1999; 96:5657-5662.
14. Curto M, Reali C, Palmieri G, Scintu F, Schivo ML, Sogos V, Marcialis MA, Ennas MG, Schwarz H, Pozzi G, et al. Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria. Neurochem Int 2004; 44:381-392.
15. Tobin DM, Roca FJ, Oh SF, McFarland R, Vickery TW, Ray JP, Ko DC, Zou Y, Bang ND, Chau TT, et al. Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections. Cell 2012; 148:434-446.
16. Allie N, Grivennikov SI, Keeton R, Hsu NJ, Bourigault ML, Court N, Fremond C, Yeremeev V, Shebzukhov Y, Ryffel B, et al. Prominent role for T cell-derived tumour necrosis factor for sustained control of Mycobacterium tuberculosis infection. Sci Rep 2013; 3:1809.
17. Francisco NM, Hsu NJ, Keeton R, Randall P, Sebesho B, Allie N, et al. TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis. J Neuroinflammation 2015; 12:125.
18. Hsu NJ, Francisco NM, Keeton R, Allie N, Quesniaux VF, Ryffel B, Jacobs, M. Myeloid and T cell-derived TNF protects against central nervous system tuberculosis. Front Immunol 2017; 8:180.
19. Hurdayal R, Ndlovu HH, Revaz-Breton M, Parihar SP, Nono JK, Govender M,  Brombacher F. IL-4-producing B cells regulate T helper cell dichotomy in type 1- and type 2-controlled diseases. Proc Natl Acad Sci USA 2017; 114:E8430-E8439.
20. Goldfeld AE, Flemington EK,  Boussiotis VA,  Theodos CM,  Titus RG,  Strominger JL, Speck SH. Transcription of the tumor necrosis factor alpha gene is rapidly induced by anti-immunoglobulin and blocked by cyclosporin A and FK506 in human B cells. Proc Natl Acad Sci USA 1992; 89:12198-12201.
21. Wilson EH, Weninger W, et al. Trafficking of immune cells in the central nervous system. J Clin Invest 2010; 120:1368-1379.
22. Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS, et al. Structural and functional features of central nervous system lymphatic vessels. Nature 2015; 523:337-341.
23. Zhang X. Regulatory functions of innate-like B cells. Cell Mol Immunol 2013; 10:113-121.
24. Buonsenso D, Serranti D, Valentini P. Management of central nervous system tuberculosis in children: light and shade. Eur Rev Med Pharmacol Sci 2010; 14:845-853.
25. Achkar JM, Chan J, Casadevall A. B cells and antibodies in the defense against Mycobacterium tuberculosis infection. Immunol Rev 2015; 264:167-181.
26. Kozakiewicz LPJ, Flynn J, Chan J. The Role of B Cells and Humoral Immunity in Mycobacterium tuberculosis Infection. In: Divangahi M. (eds) The New Paradigm of Immunity to Tuberculosis. Adv Exp Med Biol 2013; 783: 225-250.
27. Glatman-Freedman A, Casadevall A. Serum therapy for tuberculosis revisited: reappraisal of the role of antibody-mediated immunity against Mycobacterium tuberculosis. Clin Microbiol Rev 1998; 11:514-532.
28. Vordermeier HM, Venkataprasad N, Harris DP, Ivanyi J. Increase of tuberculous infection in the organs of B cell-deficient mice. Clin Exp Immunol 1996; 106:312-316.
29. Maglione PJ, Xu JY, Chan J. B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis. J Immunol 2007; 178:7222-7234.
30. Torrado E, Fountain JJ, Robinson RT, Martino CA, Pearl JE, Rangel-Moreno J, et al. Differential and site specific impact of B cells in the protective immune response to Mycobacterium tuberculosis in the mouse. PLoS One 2013; 8:e61681.
31. Phuah JY, Mattila JT, Lin PL, Flynn JL. Activated B cells in the granulomas of nonhuman primates infected with Mycobacterium tuberculosis. Am J Pathol 2012; 181:508-514.
32. Grivennikov SI, Tumanov AV, Liepinsh DJ, Kruglov AA, Marakusha BI, Shakhov AN, et al. Distinct and nonredundant in vivo functions of TNF produced by t cells and macrophages/neutrophils: protective and deleterious effects. Immunity 2005; 22:93-104.
33. Tsenova L, Ellison E, Harbacheuski R, Moreira AL, Kurepina N, Reed MB, et al. Virulence of selected Mycobacterium tuberculosis clinical isolates in the rabbit model of meningitis is dependent on phenolic glycolipid produced by the bacilli. J Infect Dis 2005; 192:98-106.
34. Tsenova L, Harbacheuski R, Sung N, Ellison E, Fallows D, Kaplan G. BCG vaccination confers poor protection against M. tuberculosis HN878-induced central nervous system disease. Vaccine 2007; 25:5126-5132.
35. Drennan MB, Nicolle D, Quesniaux VJF, Jacobs M, Allie N, Mpagi J, et al. Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection. Am J Pathol 2004; 164:49-57.
36. Scott HM, Flynn JL. Mycobacterium tuberculosis in che mokine receptor 2-deficient mice: influence of dose on disease progression. Infect Immun 2002; 70:5946-5954.
37. Francisco NM, Fang YM, Ding L, Feng S, Yang Y, Wu M, et al. Diagnostic accuracy of a selected signature gene set that discriminates active pulmonary tuberculosis and other pulmonary diseases. J Infect 2017; 75:499-510.
38. Boissel JP, Ohly D, Bros M, Godtel-Armbrust U, Forstermann U, Frank S. The neuronal nitric oxide synthase is upregulated in mouse skin repair and in response to epidermal growth factor in human HaCaT keratinocytes. J Invest Dermatol 2004; 123:132-139.
39. Castillo-Méndez SI, Zago CA, Sardinha LR, Freitas do Rosário AP, et al. Characterization of the spleen B-cell compartment at the early and late blood-stage plasmodium chabaudi malaria. Scand J Immunol 2007; 66:309-319.
40. Radwanska M, Guirnalda P, De Trez C, Ryffel B, Black S, Magez S. Trypanosomiasis-induced B cell apoptosis results in loss of protective anti-parasite antibody responses and abolishment of vaccine-induced memory responses. PLoS Pathog 2008; 4:e1000078.
41. Feng X, Yang X, Xiu B, Qie S, Dai Z, Chen K, et al. IgG, IgM and IgA antibodies against the novel polyprotein in active tuberculosis. BMC Infect Dis 2014; 14:336.
42. Jinquan T, Jacobi HH, Jing C, Millner A, Sten E, Hviid L, et al. CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells. J. Immunol 2003; 171:1722-1731.
43. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci USA 1998; 95:9448-9453.
44. Sorensen TL, Roed H, Sellebjerg F. Chemokine receptor expression on B cells and effect of interferon-beta in multiple sclerosis. J Neuroimmunol 2002; 122:125-131.
45. Forster R, Mattis AE, Kremmer E, Wolf E, Brem G, Lipp M. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell 1996; 87:1037-1047.
46. Liao F, Shirakawa AK, Foley JF, Rabin RL, Farber JM. Human B cells become highly responsive to macrophage-inflammatory protein-3α/CC chemokine ligand-20 after cellular activation without changes in CCR6 expression or ligand binding. J Immunol 2002; 168:4871.
47. Roy MP, Kim CH, Butcher EC. Cytokine control of memory B cell homing machinery. J Immunol 2002; 169:1676-1682.
48. Pellegrini A, Guinazu N, Aoki MP, Calero IC, Carrera-Silva EA, Girones N, et al. Spleen B cells from BALB/c are more prone to activation than spleen B cells from C57BL/6 mice during a secondary immune response to cruzipain. Int Immunol 2007; 19:1395-1402.
49. Yoon HS, Scharer CD, Majumder P, Davis CW, Butler R, Zinzow-Kramer W, et al. ZBTB32 is an early repressor of the CIITA and MHC class II gene expression during B cell differentiation to plasma cells. J Immunol 2012; 189:2393-2403.
50. Bosio CM, Gardner D, Elkins KL. Infection of B Cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: Delay in dissemination and development of lung pathology. J Immunol 2000; 164:6417-6425.
51. Serafini B, Rosicarelli B, Magliozzi R, Stigliano E, Aloisi F. Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis. Brain Pathol 2004; 14:164-174.
52. Magliozzi R, Howell O, Vora A, Serafini B, Nicholas R, Puopolo M, et al. Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology. Brain 2007; 130:1089-1104.
53. Taddeo A, Khodadadi L, Voigt C, Mumtaz IM, Cheng Q, Moser K, et al. Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors. Arthritis Res Ther 2015; 17:39.
54. Yi Q, Dabadghao S, Osterborg A, Bergenbrant S, Holm G. Myeloma bone marrow plasma cells: evidence for their capacity as antigen-presenting cells. Blood 1997; 90:1960-1967.
55. Vascotto F, Le Roux D, Lankar D, Faure-Andre G, Vargas P, Guermonprez P, et al. Antigen presentation by B lymphocytes: how receptor signaling directs membrane trafficking. Curr Opin Immunol 2007; 19:93-98.
56. Maglione PJ, Chan J. How B cells shape the immune response against Mycobacterium tuberculosis. Eur J Immunol 2009; 39:676-686.
57. Higuchi M,  Nagasawa K, Horiuchi T, Oike M, Ito Y, Yasukawa M, et al. Membrane tumor necrosis factor-alpha (TNF-alpha) expressed on HTLV-I-infected T cells mediates a costimulatory signal for B cell activation--characterization of membrane TNF-alpha. Clin Immunol Immunopathol 1997; 82:133-140.
58. Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNF α-deficient mice: A critical requirement for TNF α in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med 1996; 184:1397-1411.
59. Pasparakis M,  Alexopoulou L, Grell M, Pfizenmaier K, Bluethmann H, Kollias G. Peyer’s patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor. Proc Natl Acad Sci USA 1997; 94:6319-6323.
60. Clay H, Volkman HE, Ramakrishnan L. Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death. Immunity 2008; 29:283-294.
61. Roca FJ, Ramakrishnan L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species. Cell 2013; 153:521-534.
62. Qin Y, Sun X, Shao X, Cheng C, Feng J, Sun W, et al. Macrophage-microglia networks drive M1 microglia polarization after mycobacterium infection. Inflammation 2015; 38:1609-1616.
63. Achkar JM, Casadevall A. Antibody-mediated immunity against tuberculosis: implications for vaccine development. Cell Host Microbe 2013; 13:250-262.
64. Kunnath-Velayudhan S, Salamon H, Wang HY, Davidow AL, Molina DM, Huynh VT, et al. Dynamic antibody responses to the Mycobacterium tuberculosis proteome. Proc Natl Acad Sci USA 2010; 107:14703-14708.
65. Dayal R, Singh A, Katoch VM, Joshi B, Chauhan DS, Singh P, et al. Serological diagnosis of tuberculosis. Indian J Pediatr 2008; 75:1219-1221.
66. Buccheri S, Reljic R, Caccamo N, Ivanyi J, Singh M, Salerno A, et al. IL-4 depletion enhances host resistance and passive IgA protection against tuberculosis infection in BALB/c mice. Eur J Immunol 2007; 37:729-737.
67. Rodriguez A, Tjarnlund A, Ivanji J, Singh M, Garcia I, Williams A, et al. Role of IgA in the defense against respiratory infections IgA deficient mice exhibited increased susceptibility to intranasal infection with Mycobacterium bovis BCG. Vaccine 2005; 23:2565-2572.
68. Lu LL, Chung AW, Rosebrock TR, Ghebremichael M, Yu WH, Grace PS, et al. A Functional role for antibodies in tuberculosis. Cell 2016; 167:433-443 e414.