title: Lactic acid bacteria enhance autophagic ability of mononuclear phagocytes by increasing Th1 autophagy-promoting cytokine (IFN-gamma) and nitric oxide (NO) levels and reducing Th2 autophagy-restraining cytokines (IL-4 and IL-13) in response to Mycobacterium tuberculosis antigen.
abstract: OBJECTIVE
  Control of the intracellular Mycobacterium tuberculosis (Mtb), mainly requires an appropriate ratio of Th1/Th2 cytokines to induce autophagy, a physiologically, and immunologically regulated process that has recently been highlighted as an innate defense mechanism against intracellular pathogens. Current vaccines/adjuvants induce both protective Th1 autophagy-promoting cytokines, such as IFN-gamma, and immunosuppressive Th2 autophagy-restraining cytokines, such as IL-4 and IL-13. TB infection itself is also characterized by relatively high levels of Th2 cytokines, which down-regulate Th1 responses and subsequently subvert adequate protective immunity, and a low ratio of IFN-gamma/IL-4. Therefore, there is a need for a safe and non-toxic vaccine/adjuvant that will induce Th1 autophagy-promoting cytokine (IFN-gamma) secretion and suppress the pre-existing subversive Th2 autophagy-restraining cytokines (IL-4 and IL-13). As lactic acid bacteria (LAB) belonging to the natural intestinal microflora and their components have been shown to shift immune responses against other antigens from Th2-type cytokines toward Th1-type cytokines like IFN-gamma, we investigated whether LAB can improve the polarization of Th1/Th2 cytokines and autophagic ability of mononuclear phagocytes in response to Mtb antigen.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMCs), which are a part of the mononuclear phagocyte system and source of crucial macrophage activators in the in vivo situation, and human monocyte-derived macrophages (HMDMs) were treated with Mtb antigen in the presence or absence of two strains of LAB, L. rhammosus GG (LGG) and Bifidobacterium bifidum MF 20/5 (B.b). PBMCs cell culture supernatants were analyzed for the production of the autophagy-promoting factors IFN-gamma, and nitric oxide (NO) and the autophagy-restraining cytokines IL-4 and IL-13, using ELISA and Griess assays to detect the production of cytokines and NO, respectively. In HMDMs, expression of microtubule-associated protein 1 light chain 3 (LC3-I), membrane-associated (LC3-II) forms of LC3 protein and Beclin-1, as hallmarks of autophagy, were assessed using Western blot to detect the autophagy markers. The secreted interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin (IL)-12 and transformig growth factor-beta (TGF-beta), and chemokine (C-C motif) ligand 18 (CCL18) from HMDMs were determined by ELISA. Also, reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the mRNA expressions of CCL18 in HMDMs.
  
  
  RESULTS
  Treatment of PBMCs with either Mtb antigen or with LAB significantly increased the IFN-gamma and NO production. Combination of Mtb antigen and LAB led to synergistic increase in IFN-gamma, and an additive increase in NO. Treatment with Mtb antigen alone significantly increased the IL-4 and IL-13 production. LAB significantly decreased IL-4 and IL-13 secretion in both unstimulated and Mtb antigen-stimulated PBMCs. The IFN-gamma/IL-4+IL-13 ratio was enhanced, indicating Th1/Th2 polarization. Treatment of macrophages with combined use of Mtb antigen and LAB led to an additive increase in Beclin-1, LC3-II expression, as well as in synergistic increase in IL-12 production. Treatment of macrophages with combined use of Mtb antigen and LAB led to a decrease in IL-6, IL-10, and CCL18 secretion. LAB inhibited the secretion of TGF-beta by Mtb-stimulated macrophages, however not significantly. Treatment of macrophages with combined use of Mtb antigen and LAB led to a decrease in CCL18 mRNA expression.
  
  
  CONCLUSIONS
  Our study implies that LAB may reinforce the response of the mononuclear phagocytes to Mtb antigen by inducing production of the autophagy-promoting factors IFN-gamma and NO, while decreasing the Th2 autophagy-restraining cytokines IL-4 and IL-13. Hence, combination of Mtb antigen and LAB may perhaps be safer in more efficacious TB vaccine formulation.
authors.affiliation: Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut, Hermann-Weigmann-Str 1, D-24103 Kiel, Germany. ghadimi@bafm.de
authors.forename: Darab
authors.initials: D
authors.lastname: Ghadimi
authors.forename: Michael
authors.initials: M
authors.lastname: de Vrese
authors.forename: Knut J
authors.initials: KJ
authors.lastname: Heller
authors.forename: Juergen
authors.initials: J
authors.lastname: Schrezenmeir
mesh_terms: D000328:Adult; D000942:Antigens, Bacterial / Q000276:immunology*; D001343:Autophagy / Q000276:immunology*; D001644:Bifidobacterium / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology*; D018793:Interleukin-13 / Q000032:analysis / Q000378:metabolism; D015847:Interleukin-4 / Q000032:analysis / Q000378:metabolism; D052201:Lacticaseibacillus rhamnosus / Q000276:immunology* / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000276:immunology; D009000:Monocytes / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000378:metabolism; D032581:Tuberculosis Vaccines / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D018793:Interleukin-13; D008869:Microtubule-Associated Proteins; D016212:Transforming Growth Factor beta; D032581:Tuberculosis Vaccines; C072681:Mycobacterium tuberculosis antigens; D015847:Interleukin-4; D009569:Nitric Oxide; D007371:Interferon-gamma