The Host Response to a Clinical MDR Mycobacterial Strain Cultured in a Detergent-Free Environment: A Global Transcriptomics Approach

Abstract

During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in infection experiments.

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…We were however unable to detect ESAT6 in infected macrophages by immunolabeling, although ESAT6 clearly was present on bacilli before contact with macrophages. Our study as well as findings from others warrant a re-evaluation of cultivation conditions for experimental studies, since the sole presence of a mycobacterial capsule elicited differential responses in macrophages and in a murine vaccination model, and cultivation without detergent profoundly altered the transcriptome of Mtb [356,359].…”

“…Striking differences in the transcriptional response of primary murine macrophages to an MDR strain cultivated in the presence and absence of detergent were found [356]. Mtb devoid of a capsule induced a strong pro-inflammatory response, and downregulated for example the expression of the IFNγ receptor, which could promote survival and persistence of Mtb [356,357].…”

“…Striking differences in the transcriptional response of primary murine macrophages to an MDR strain cultivated in the presence and absence of detergent were found [356]. Mtb devoid of a capsule induced a strong pro-inflammatory response, and downregulated for example the expression of the IFNγ receptor, which could promote survival and persistence of Mtb [356,357]. Another study looking into the transcriptional response of bovine alveolar macrophages after M. bovis infection obtained conclusions different from other studies with similar settings, partly attributed to the use of detergent-free cultivated bacilli, but unfortunately a direct comparison of the transcriptomics upon infection of Mtb cultivated with or without detergent was not performed here [358].…”

“…ESAT6, CFP10 and other ESX-1 substrates have been suggested to reside in the mycobacterial capsule [35,38], and so far conflicting evidence exists whether encapsulated Mtb elicit stronger pro-inflammatory responses in macrophages than non-encapsulated bacilli [38,356].…”

Trick or tween: An inflammatory surprise when M. tuberculosis knocks a cell's door and no tween is provided

“…15 Tween 80 is a detergent commonly added into the growth medium of M. tuberculosis to prevent clumping, which prevents accurate determination of the ratio of the infecting bacteria number to the number of targeted host cells. Therefore, it is a common practice to include Tween 80 when preparing M. tuberculosis for infections, which was the case in all previous transcriptional response studies.…”