An integrated omics analysis: impact of microgravity on host response to lipopolysaccharide in vitro

Abstract

Background: Microgravity facilitates the opportunistic infections by augmenting the pathogenic virulence and suppressing the host resistance. Hence the extraterrestrial infections may activate potentially novel bionetworks different from the terrestrial equivalent, which could only be probed by investigating the host-pathogen relationship with a minimum of terrestrial bias.

Results: We customized a cell culture module to expose human endothelial cells to lipopolysaccharide (LPS). The assay was carried out onboard the STS-135 spaceflight, and a concurrent ground study constituted the baseline. Transcriptomic investigation revealed a possible immune blunting in microgravity suppressing in particular Lbp, MyD88 and MD-2, which encode proteins responsible for early LPS uptake. Certain cytokines, such as IL-6 and IL-8, surged in response to LPS insult in microgravity, as suggested by the proteomics study. Contrasting proteomic expressions of B2M, TIMP-1 and VEGRs suggested impaired pro-survival adaptation and healing mechanisms. Differential expression of miR-200a and miR-146b suggested the susceptibility of hosts in spaceflight to oxidative stress and further underscored the influence of microgravity on the immunity.

Conclusions: A molecular interpretation explaining the etiology of the microgravitational impact on the host-pathogen relationship elucidated comprehensive immune blunting of the host cells responding to LPS challenges. Longer LPS exposure prompted a delayed host response, potentially ineffectual in preventing pathogens from opportunistic invasion. Significant consequences include the subsequent failure in recruiting the growth factors and a debilitated apoptosis. Follow up studies with larger sample size are warranted.

Figure 1

Experiment scheme followed in the spaceflight and the concurrent ground study . Briefly, each of the two CCMs were loaded with six bioreactors. One CCM was operated in the spaceflight and the other one on ground, concurrently. Both CCMs followed same protocol depicted in the figure; where the top row shows the overall assay sequence segregated into three time zones: (i) the first 10 days, (ii) 11^th day and (iii) the last 5 days of the space mission. The protocol carried out on the 11^th day is elaborated inside the box bordered by the broken lines. The cells received typical nourishments during first 10 days of space mission. At the beginning of the 11^th day, 100 μg/ml LPS were injected to the flowpaths of bioreactors 5 and 6 in both CCMs (arrowhead with LPS 0 h). Four hours later, the same amount of LPS was injected into bioreactor 3 and 4 (arrowhead with LPS 4 h); and after the subsequent four hours, RLT solution was discharged, replacing the media in the flowpaths of all bioreactors [–6] (arrowhead with RLT 8 h). The biosamples in the bioreactors [–6] in both gravity limits were preserved in the RLT solution till the space shuttle landed 5 days after the RLT discharge. We collected the bioreactors 3 hours after the landing.

Figure 2

Principal component analysis (PCA) of 5,379 transcriptomic signatures of host responses to LPS assault mediated by different gravitational forces. The arrows indicate the shifts of the transcriptomic expressions due to LPS insults at two gravitational limits. The genes were mined by pair-wise comparison between the LPS-treated samples normalized by the untreated samples inoculated on ground and in spaceflight respectively; for instance, G-4 h and G-8 h were normalized by G-C and S-4 h and S-8 h were normalized by S-C. The close Euclidian proximity of the self-normalized G-C and S-C implies that variance profile of this PCA does not represent the exclusive impact of microgravity. A distant clustering between the LPS assault carried out on ground (G-4 h and G-8 h) and that carried out in spaceflight, S-4 h in particular, is marked by PC 1 explaining 80% of total variance. Thereby the impact of microgravity on LPS assault emerges as the most significant factor in explaining the genomic perturbation among these 5,379 genes. Modest Euclidian proximity between S-4 h and S-C (r = 0.16) suggests an immune blunting in spaceflight. Clearly, the host in spaceflight failed to respond to LPS assault. The position of S-8 h equidistant from the two clusters formed by (a) S-C, G-C and S-4 h and to (b) G-4 h and G-8 h suggests a delayed host response in spaceflight.

Figure 3

Enrichment profile of the immunologically relevant pathways significantly altered by GoI-LPSμG with a focus on Toll-like receptor (TLR) pathway co-enriched by GoI-LPSμG and GoI-μG. A. Enrichment profile of the immunologically relevant pathways significantly altered in responding to LPS assault mediated by different gravitational forces. Twenty four most significantly enriched pathways were identified focusing on three parent nodes, namely cytokine/chemokine pathways (11 networks) apoptosis pathways (5 networks) and growth factor pathways (8 networks). For each network, there are four bars segregated into two columns (Ground and Space) and two rows (4 h and 8 h, which are noted as 4 and 8, respectively). The distributions of genomic regulations associated with each pathway are hereby documented for G-4 h, G-8 h, S-4 h and S-8 h and the pathways are batched into three parent pathways: cytokine/chemokine pathways, apoptosis pathways and growth factor pathways. The fraction of the elevated (fold change > 2) and suppressed (fold change < −2) transcripts are colored by red and green, respectively. The unchanged fractions are yellow. The color scheme is at the right. B. Toll-like receptors (TLR) pathway. A schematic network of TLRs pathway is presented here. The TLRs pathway is significantly co-enriched by both gene sets linked to LPS insult mediated by microgravity (GoI-LPSμG) and exclusive effects of microgravity (GoI-μG), respectively. There are three blocks beneath each gene name representing the transcriptomic regulations of S-C (left most block), G-4 h (middle block) and S-4 h (right most block). G-8 h and S-8 h are omitted. The S-C regulations normalized by G-C (S-C/G-C) represent the exclusive impacts of microgravity. And, G-4 h and S-4 h normalized by respective controls (G-4 h/G-C and S-4 h/S-C) represent the influence of the gravitational shift on host response to LPS assault. The scale of the color scheme is included.

Figure 4

miRNA signatures of host responses to LPS assault mediated by different gravitational forces and corresponding mRNA targets. Fifteen miRNAs significantly altered by 4 h LPS exposures carried out in two gravitational limits are clustered in two middle columns. There are 398 mRNAs collectively targeted by 15 miRNAs of interest. The four columns from left to right indicate the mRNA targets (1^st column) and corresponding miRNA probes (2^nd column), both regulated by the LPS assault in terrestrial gravity; miRNA probes (3^rd column) and corresponding mRNA targets (4^th column) both regulated by LPS assault in μG. The mRNAs are batched according to their miRNA modulators. The scale representing the color scheme is included.

Figure 5

Proteomics profile illustrating the impact of microgravity on the immune responses to LPS assault. The hierarchical clustering depicts the proteomic expression of the focused cytokine profile sampled from the 4 h LPS exposures carried out in two gravitational limits. The columns from left to right indicate the proteomic expression observed (i) in spaceflight compared to ground control (S-C/G-C)- marked as S-C, (ii) LPS exposure for 4 h compared to ground control (G-4 h/G-C)- marked as G-4 h and (iii) LPS exposure for 4 h compared to space control (S-4 h/S-C)- marked as S-4 h. The scale representing the color scheme is included.