Mycobacterium tuberculosisstimulates IL-1β production by macrophages in an ESAT-6 dependent manner with the involvement of serum amyloid A3

Abstract

TX 75708-3154, phone 29 (903)-877-7665, fax (903) 877-5516, buka.samten@uthct.edu 30 4. Acknowledgment: We thank Dr. Charles A. Dinarello for his thoughtful guidance and 31 discussions.32 5. Brief summary: Mycobacterium tuberculosis stimulated the production of IL-1β by 33 macrophages and in mouse lungs with the activation of NLRP3 and K+ efflux in an ESAT-6 34 dependent manner with the involvement of SAA3. 35 36 37 38 39 40 41 42 43 44 45 46 3 ABSTRACT 47To explore interleukin (IL)-1β production in tuberculosis, we infected mouse bone marrow-derived 48 macrophages (BMDM) with Mycobacterium tuberculosis (Mtb) H37Rv, its early secreted antigenic target 49 protein of 6 kDa (ESAT-6) gene deletion (H37Rv:∆3875) or complemented strain (H37Rv:∆3875C) and 50 evaluated IL-1β production. H37Rv induced significantly increased IL-1β production by BMDMs 51 compared to non-infected BMDMs. In contrast, H37Rv:∆3875 induced significantly less mature IL-1β 52 production despite eliciting comparable levels of pro-IL-1β and IL-8 from BMDMs compared to H37Rv 53 and H37Rv:∆3875C. Blocking either NLRP3 or K + efflux diminished H37Rv-induced IL-1β production 54 by BMDMs. Infection of mice intranasally with H37Rv:∆3875 induced less IL-1β production in the lungs 55 compared with H37Rv.Intranasal delivery of ESAT-6 but not CFP10 induced production of IL-1β in 56 mouse lungs and RNA-Seq analysis identified serum amyloid A (SAA) 3 as one of the highly expressed 57 genes in mouse lungs. Infection of mice with H37Rv but not H37Rv:∆3875 induced expression of lung 58 SAA3 mRNA and protein, consistent with the effect of intranasal delivery of ESAT-6. Silencing SAA3 59 reduced Mtb-induced IL-1β production by BMDMs. We conclude that the production of SAA3 is required 60 for Mtb stimulated IL-1β production by macrophages in tuberculosis infection. 61 62 63 64 65 66 67 68 69 70Tuberculosis (TB) caused around 1.2 million deaths among HIV-negative people and an additional 71 251,000 deaths among HIV-positive people in 2018 (1), which is mainly due to the lack of a detailed 72 understanding of the molecular mechanisms of interactions between immune cells and the pathogen, 73 Mycobacterium tuberculosis (Mtb). Improved understanding of the host-pathogen interaction will lead to 74 an effective TB vaccine design and an identification of novel drug targets to improve clinical management 75 of drug-resistant TB infections. Macrophages play critical roles in tuberculosis infection as both host cells 76 providing intracellular niche for Mtb infection and growth and effector immune cells fighting against Mtb 77infection by communicating with other immune effector cells by producing different cytokines (2). 78Although IL-1β production by macrophages is pivotal for protection against TB infection (3, 4), several 79 clinical reports showed that IL-1β is also involved in TB pathogenesis. There is significantly increased 80 IL-1β mRNA in the alveolar macrophages and IL-1β protein in bronchoalveolar lavage fluid of TB 81 patients compared with healthy controls (5...

Interactions between CNS and immune cells in tuberculous meningitis

“…Lee et al have revealed that the activation of primary murine microglia in conditioned media from M.tb-infected macrophages was associated with the maturation of caspase-1 and IL-1b in microglia during the M.tb infection (109). Generally, M.tb could be maintained in a silencing status by regulating the expression of interleukins in the homeostatic immune response (154,155). Moreover, signal transducer and activator of transcription 1 (Stat1) and interferon regulatory factor1 (IRF1) have been verified to be involved in the inflammation response of macrophages and microglia after TBM induced by attenuated M.tb (74).…”

Impaired expression of the TWIK2 K+ efflux channel leads to reduced IL-1 family cytokine production in patients with nontuberculous mycobacterial lung disease

“…Pharmacological inhibition of TLR2 and TWIK2 C29 (MedChemExpress) and quinine (Sigma) were used as TLR2-selective inhibitors (20) and TWIK2selective inhibitors (21,22), respectively. PBMCs from healthy volunteers were stimulated with 10 µg/ml heat-killed MAC antigens in the presence or absence of various concentrations (10, 50 and 100 µM) of C29 or quinine at 37 °C in 5% CO 2 for 24 h. CD14+ monocytes from healthy volunteers were stimulated with live MAC (MOI of 10) in the presence or absence of various concentrations (10, 50 and 100 µM) of C29 or quinine at 37 °C in 5% CO 2 for 18 h. The cell-free supernatants were collected and stored at -80 °C until use.…”

“…TLR2, P2X7R and two-pore domain potassium (K+) e ux channels are involved in mycobacteria-induced proin ammatory signaling, especially IL-1β production (22,26,27). To determine whether any of these receptors and channels are involved in reduced IL-1β, IL-18, IL-1α and IL-10 production, we cultured PBMCs of NTMPD patients and their healthy household contacts with or without heat-killed M. intracellulare as mentioned in the methods section.…”