Evaluating the Effect of Spaceflight on the Host–Pathogen Interaction

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BRIEF COMMUNICATION OPEN Evaluating the effect of spaceﬂight on the host–pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium

Jennifer Barrila 1,2, Shameema F. Sarker2, Nicole Hansmeier3,4, Shanshan Yang5, Kristina Buss5, Natalia Briones5,6, Jin Park5, Richard R. Davis1,2, Rebecca J. Forsyth2,16, C. Mark Ott7, Kevin Sato8, Cristine Kosnik9, Anthony Yang9, Cheryl Shimoda9, Nicole Rayl8,10, Diana Ly8, Aaron Landenberger11,16, Stephanie D. Wilson12, Naoko Yamazaki13, Jason Steel5, Camila Montano5, Rolf U. Halden14, ✉ Tom Cannon9, Sarah L. Castro-Wallace7 and Cheryl A. Nickerson 1,2,15

Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host–pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells. npj Microgravity (2021) 7:9 ; https://doi.org/10.1038/s41526-021-00136-w 1234567890():,;

Humans will travel to space more frequently and for longer and their external environment to regulate cellular responses17–19. duration as the rapidly growing spaceflight industry enables more Since spaceflight alters the organization and/or expression of routine access for both professional astronauts and the public. these classes of proteins14,20,21, it is logical to predict that this Spaceflight can adversely affect multiple aspects of human environment could impact infection processes for invasive physiology, including immune and gastrointestinal function1–4. pathogens like Salmonella that hijack the host and actively exploit While pre-flight screening and quarantine procedures have these structural networks to enable their invasion and reduced infection incidence, astronauts and cargo can still harbor dissemination22,23. opportunistic and obligate pathogens, including Salmonella spp., From the pathogen perspective, we previously discovered that which have been recovered from both crew refuse and Interna- spaceflight analog culture of Salmonella in the Rotating Wall tional Space Station (ISS) surfaces5–8. We previously discovered Vessel bioreactor led to increased virulence in mice24, enhanced – that spaceflight and spaceflight analog culture increased the stress resistance9 11,24, global transcriptional and proteomic virulence and stress resistance of the foodborne pathogen, reprogramming11,12,24 and enhanced the colonization of a human Salmonella enterica serovar Typhimurium (Salmonella)9–12. These 3D intestinal co-culture model (JB, SY, RRD, RJF, CMO, JS, CAN; environments have also been shown to increase the virulence manuscript in preparation). Space Shuttle experiments aboard and/or alter pathogenesis-related phenotypes of other micro- STS-115 and STS-123 independently confirmed RWV trends by organisms13–16. Collectively, these findings indicate an increased demonstrating that spaceflight increased the virulence and risk for infection during space travel and underscore the need for globally altered the transcriptomic and proteomic responses of a more in-depth understanding of the similarities and differences Salmonella relative to identical ground controls9,10. Supplementa- between spaceflight and ground-based infection mechanisms. tion of select ions in the media was able to counteract this Studies performed in spaceflight and spaceflight analog spaceflight-associated increased virulence10. It is important to environments have revealed unexpected physiological responses note that Salmonella virulence studies were performed when the in both mammalian and microbial cells14,15. This is not surprising bacteria were cultured in spaceflight, brought back to Earth and given that physical forces in the cellular microenvironment immediately used in animal infections (i.e., the host was not (including those associated with microgravity) profoundly alter exposed to spaceflight). Transcriptional profiling revealed a central both host and pathogen phenotypes through mechanotransduc- role for the RNA-binding protein Hfq in the response of Salmonella tion17. An integrated network involving the cytoskeleton, cell to both spaceflight and spaceflight analog culture in the RWV9,10 surface receptors, and the extracellular matrix (ECM) mediates the and subsequent studies indicated altered expression of hfq or the dynamic three-dimensional (3D) interactions between host cells Hfq regulon in other microbes25–28.

1Biodesign Center for Fundamental and Applied Microbiomics, Arizona State University, Tempe, AZ, USA. 2Biodesign Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ, USA. 3Swette Center for Environmental Biotechnology, The Biodesign Institute, Arizona State University, Tempe, AZ, USA. 4Luther College at University of Regina, Department of Biology, Regina, Saskatchewan, Canada. 5Bioinformatics Core Facility, Bioscience, Knowledge Enterprise, Arizona State University, Tempe, AZ, USA. 6Integrated Cancer Genomics Division, The Translational Genomics Research Institute, Phoenix, AZ, USA. 7Biomedical Research and Environmental Sciences Division, NASA Johnson Space Center, Houston, TX, USA. 8NASA Ames Research Center, Mountain View, CA, USA. 9Tissue Genesis, Inc, Honolulu, HI, USA. 10NASA Headquarters, Washington, D.C., USA. 11DoD Space Test Program, Houston, TX, USA. 12Astronaut Ofﬁce, NASA Johnson Space Center, Houston, TX, USA. 13Space Biomedical Research Ofﬁce, Human Space Technology and Astronauts Department, Japan Aerospace Exploration Agency (JAXA), Tokyo, Japan. 14Biodesign Center for Environmental Health Engineering, School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ, USA. 15School of Life Sciences, Arizona State University, Tempe, AZ, USA. 16Deceased: ✉ Rebecca J. Forsyth, Aaron Landenberger. email: [email protected]

Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Barrila et al. 2 In this study, we investigated how spaceflight culture alters the limitations precluded the use of gentamicin protection assays to response of human intestinal epithelial cells (HT-29) to infection by quantify bacterial invasion into the epithelium, we focused our Salmonella when both the host and pathogen were exposed to analysis on transcriptomic (dual RNA-seq of host and pathogen) spaceflight. The experiment was flown as part of the Space Tissue and proteomic profiling (Isobaric tags for relative and absolute Loss (STL) payload and operationally designated as STL-IMMUNE, quantification/iTRAQ). These global analyses can yield valuable since epithelial cells are excellent sentinels of innate immunity by insight into the status of healthy and infected cells. Figure 1 shows providing the first physical and chemical barriers to infection and the experimental design and timeline for STL-IMMUNE and coordinating additional innate and mucosal defenses and sub- schematic of hollow fiber bioreactors used for 3D cell culture. sequent adaptive responses29,30. Since in-flight technical HT-29 cells were seeded into the extracapillary space (ECS) of 1234567890():,;

Fig. 1 STL-IMMUNE experimental design and impact of spaceflight on uninfected HT-29 transcriptional profiles. a Hollow fiber bioreactor technology. Photo and cross-sectional schematic of the hollow fiber bioreactors used in this study. Hollow fibers had a pore size of 0.3 µm to retain bacteria within the extracapillary space (ECS) while still facilitating nutrient and gas exchange via diffusion as oxygenated media was perfused through the intracapillary space (ICS). b STL-IMMUNE experimental design. HT-29 cells and bacteria were prepared as shown (see “Methods” section for additional detail). Bioreactors, bacteria, and fixative bags were integrated into the CCM hardware and launched aboard Space Shuttle Discovery mission STS-131. Crew members activated the CCM once on orbit. At mission elapsed day 11, cultures were infected with S. Typhimurium for 6 h and fixed for postflight analysis. Launch and astronaut images: NASA [STS-131-S-036 (image is slightly cropped), S131E007206, and S131E007188]. c Spaceflight-induced transcriptional responses of HT-29 cells relative to ground controls. Left panel: volcano plot depicting differentially expressed genes (DEGs) between flight and ground control cultures (flight/ground). Red: significantly upregulated genes according to a log2 fold change in expression of at least ± 1 and FDR < 0.05. Blue: significantly downregulated genes. The top 5 significantly up and downregulated genes are labeled. Right panel: Analysis of upregulated (red) and downregulated (blue) transcripts revealed significantly enriched GO terms and KEGG pathways using DAVID. BP biological process, CC cellular component. The authors affirm that informed consent for publication of the images in Fig. 1 was provided.

npj Microgravity (2021) 9 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Barrila et al. 3 hollow fiber bioreactors which contained collagen I-coated metabolic plasticity to exploit diverse nutrients in parallel, even capillary fibers for intestinal cell attachment. Oxygenated and at trace levels44. CO2-containing media was continually perfused through the Engagement of IFN-I signaling and decreased expression of intracapillary space (ICS) of the fibers (underneath the basal side metabolic genes could also indicate increased apoptosis. How- of the cells, which also prevented exposure of cells to high fluid ever, pro-apoptotic genes BNIP3 and BNIP3L were downregulated shear forces). During spaceflight, Salmonella was introduced to the in flight, suggesting reduced apoptosis. A previous spaceflight apical side of HT-29 cultures through ECS ports and allowed to study45 showed reduced apoptosis and increased differentiation incubate for 6 h before fixative was perfused through the ICS. in MIP-101 colon cells, as well as increased expression of Between uninfected flight and ground HT-29 cultures, RNA-seq carcinoembryonic antigen proteins (often upregulated in colon profiling showed significant differences in 307 genes (≥2-fold cancer). In our study, we observed the downregulation of change; false discovery rate (FDR) < 0.05; Fig. 1c and Supplemen- CEACAM6 (Supplementary Table 1). IFN-I signaling can also be tary Table 1). Evaluation of the top 5 significantly upregulated activated by the DNA damage response following exposure to genes revealed the enhanced expression of several transcripts radiation, oxidative stress, or other harmful stimuli. DNA damage belonging to the small proline-rich (SPRR) family as well as DHRS9 can occur during spaceflight due to radiation exposure and (a retinol dehydrogenase) and tumor-associated calcium signal oxidative stress, including in the gastrointestinal tract46–49. transducer 2 (Fig. 1c). SPRR genes encode mechanosensitive Chromosomal damage has been observed in astronauts48,49 and proteins that play roles in barrier function, differentiation, reactive we previously saw the downregulation of select DNA repair genes oxygen species (ROS) detoxification, wound healing and cancer31. in astronaut blood cells50, which could potentially impede repair. They are also likely involved in the stabilization of the actin In our present study, several genes involved in checkpoint- cytoskeleton in response to biomechanical stress32. Their expres- mediated responses to DNA damage and replication stress were sion is regulated by ECM/integrin interactions and by various upregulated (BRCA1, CLSPN, MCM10, RRM2, and KIAA0101). Thus, it stimuli, including UV radiation, inflammation, oxidative stress, is also possible that changes in IFN-I and metabolic genes could DNA damage, and retinoids31,33. Gene ontology (GO) analysis of be due to DNA damage caused by oxidative stress and/or upregulated genes indicated enrichment in type I interferon (IFN-I) radiation exposure. signaling/antiviral defense, DNA replication, and differentiation GO analysis of downregulated genes indicated enrichment in (including the SPRR genes) (Fig. 1c, Supplementary Table 2). The cellular components related to the cell surface/membrane and upregulation of viral defense genes was also previously observed extracellular components as well as complement and coagulation in the livers of mice following spaceflight34. pathways (Fig. 1c, Supplementary Table 2). Decreased expression IFN-I signaling is complex and context dependent, regulating a of genes encoding factors classically associated with blood or broad range of cellular processes35,36. In addition to modulating other tissues was observed, including complement and fibrinogen innate and adaptive immune defenses against pathogens, IFN-I complex proteins (e.g., C3, FGG, FGA, and FGB) (Supplementary regulates intestinal homeostasis, including epithelial cell prolifera- Table 2). Fibrinogen and complement proteins are also expressed tion, and barrier function36,37. IFN-I signaling is often associated by epithelial cells and play an important role in infection51–54. with decreased proliferation to promote differentiation, cellular However, as there are cases where RNA from fetal bovine serum repair, or defense against infection36,37. We found that genes (FBS) in culture media has been detected in mRNA analyses55,we important for glycolysis (SLC2A3, HK2, PFKFB3, PFKFB4, PDK1) and cannot exclude the possibility of FBS interference. Importantly, the fatty acid metabolism (FADS1, FADS2, ELOVL5, EHHADH, SIRT4) same batch of heat-inactivated FBS was used for identical and were downregulated in flight (Supplementary Table 1). Previous simultaneous preparation of all flight and ground cultures in spaceflight studies using either cultured macrophages38 or murine this study. liver tissue34 also saw the suppression of multiple glycolytic We also performed iTRAQ analysis to obtain a snapshot of the enzymes, with the latter study postulating a model linking cellular proteomic profile in the uninfected flight cultures spaceflight changes in cellular metabolism, ROS, and immune compared to ground controls. The presence of FBS in the culture dysregulation34. The downregulation of these bioenergetic path- media resulted in strong interference from bovine proteins; a ways in our study could indicate the reduced proliferation of the subset of which could not be distinguished from human proteins flight cultures, which could result from a number of phenotypes, due to their identical sequence homologies and thus were including decreased tumorigenicity/increased differentiation, excluded from our analysis. Despite this challenge, we observed increased DNA damage or apoptosis. During cancer metabolism, flight-associated upregulation of the major cytoskeletal proteins glycolysis and fatty acid synthesis is often elevated39. Several G-actin and tubulin, which are known targets of Salmonella genes highly expressed in tumors or undifferentiated cells were effectors to facilitate invasion56 (Supplementary Table 3). HSPA1A also downregulated in flight, including CA9, CA12, CEACAM6, protein was also 2.4-fold upregulated in flight. None of these ITGA5, CLDN2, COL5A2, VIM, PROM1, and LGR4 (Supplemental Table proteins were differentially regulated at the transcriptional level; 1). Together, these trends may indicate decreased tumorigenicity however, it is fairly common to see divergence in mRNA and of the flight cells. Salmonella is known to preferentially colonize protein expression due to a variety of biological factors, such as cancer cells40, thus differences in tumorigenicity between flight alterations in posttranscriptional modifications, translation effi- and ground cultures could impact infection. Of relevance, we ciency and RNA or protein stabilities (reviewed by Maier et al.)57. previously showed that RWV culture of HT-29 cells induced a more We then examined the data for the infections performed with normal, differentiated phenotype relative to controls41. Although Salmonella during spaceflight and in our ground controls. The in our present study we saw indicators of increased differentiation experiment was designed to automatically inject 1 mL bacterial of the flight cultures, there were also some potential indicators of inoculum (~1 × 107 colony forming units (CFU)) into HT-29 increased tumorigenicity (e.g., downregulation of VIL, TP53I11, cultures. Postlanding hardware inspection revealed that all flight upregulation of CCAT1; Supplemental Table 1). In addition, LGR4 is samples received the intended volume of bacteria; however, also important for Paneth cell differentiation42. A reduction in ground controls received slightly different volumes (see “Meth- Paneth cells could diminish antimicrobial defenses. Host metabo- ods” section). The 6-h incubation in the absence of gentamicin lism is also important in the context of an impending infection as would have allowed sufficient time for the bacteria to replicate in the pathogen must scavenge for available nutrients43,44. the media during attachment and invasion, but it is unknown Decreases in intracellular glucose could dampen intracellular whether differences in initial inoculum eventually equalized. In pathogen replication depending on the availability of other addition, while we had sufficient material to perform iTRAQ nutrients. However, Salmonella has evolved an impressive analysis using triplicate cultures, only one infected flight sample

Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 9 J. Barrila et al. 4 had enough material remaining after proteomics for RNA-seq, by sequestering NF-κB in the cytoplasm to prevent its transloca- thus we analyzed multiple technical replicates from a single flight tion to the nucleus62. RelB is induced in response to severe bioreactor for that condition (see “Methods” section). Acknowl- systemic inflammation, LPS, flagellin and/or TNF stimulation and is edging these limitations, given the rare nature of these samples required for sustained expression of IκBα during LPS tolerance62. due to the inherent difficulty in performing spaceflight infections, Both proteins are known to be targeted by Salmonella to suppress we present a preliminary analysis of the host and pathogen host inflammation and/or apoptosis63,64. Changes in the expres- response during spaceflight infection. Rather than focusing on the sion of these transcripts may reflect an attempt by the host and/or direct quantitative comparison between the infected flight and pathogen to initiate processes to suppress excessive inflammation ground cultures, we instead focused on evaluating the gene or apoptosis during the flight infection. expression patterns elicited during either a spaceflight or ground- Ground cultures uniquely displayed enrichment in select based infection relative to their respective controls (infected vs. upregulated genes associated with cell chemotaxis and inflamma- uninfected for flight and ground) (Fig. 2; Supplementary Tables 4– tion (the latter include genes classified by DAVID under the 7). However, we also include the direct comparisons of the host ‘Rheumatoid arthritis’ category but overlap in their role for response to infection between flight and ground for comparison Salmonella infection). Conversely, downregulated genes were in future studies (Supplementary Table 8), as well as the bacterial enriched for diverse biological processes (e.g., cell adhesion, genes expressed above background during the flight and ground ECM organization, fibrinogen complex, glycolysis, hypoxia), infections (Supplementary Tables 9 and 10) and directly between cellular components (e.g., cell surface/membrane, exosome, and flight and ground infected cultures (Supplementary Table 11). fibrinogen complex), molecular functions associated with binding Analysis of flight and ground cultures indicates both common and KEGG pathway involved in ECM-receptor interaction. Inter- and distinct host transcriptional profiles in response to Salmonella estingly, although we observed the expected stimulation of TLR4 infection (Fig. 2; Supplementary Tables 4–7). Overall, 35 transcripts (Toll-like receptor 4) in the ground-based infection (Supplemen- were altered in response to infection in both flight and ground tary Table 6), this did not occur in the flight cultures at the time conditions; 28 genes in the same direction and 7 oppositely point evaluated. In alignment with the TLR4 trends, LPS-inducible regulated (Fig. 2b, Supplementary Tables 4–7). Relative to their PIGR was also upregulated only during the ground infection respective uninfected controls, both flight- and ground-infected (Supplementary Table 6). A previous study using astronaut cultures displayed enrichment in biological functions and path- monocytes observed a significant decrease in TLR4 expression ways expected for Salmonella-infected cultures, including the immediately following return from a space shuttle mission relative upregulation of genes involved in inflammation (TNF, IL-1, NF-κB, to measurements taken 10 days before launch and 15 days post and NOD signaling), response to lipopolysaccharide (LPS), and cell landing65. Similarly, TLR4 was also downregulated in blood cells in- chemotaxis (Fig. 2d; Supplementary Tables 5 and 7). Interaction of flight and post-flight relative to pre-flight levels during the Twins Salmonella with the intestinal epithelium initiates a series of study48. PIGR encodes polymeric immunoglobulin receptor which signaling cascades leading to intestinal inflammation58. In vivo, transports dimeric secretory IgA (sIgA) across the epithelium and this defensive response serves to recruit neutrophils and other into intestinal secretions as part of the first line of defense in immune cells to the site of infection but can also lead to the mucosal immunity. Our findings could indicate the potential for an generation of ROS that confers a strategic advantage for altered intestinal mucosal immune response to infection in Salmonella to outcompete resident microbiota58. Both flight and spaceflight. Hindlimb unloading disrupted intestinal immune ground infections induced upregulation of IRAK2, a protein that homeostasis in rats, leading to decreased sIgA levels in the ileum, plays a central role in TLR signaling to NF-κB for the production of although the activation of TLR4/MyD88/NF-kB was still observed66. pro-inflammatory mediators59. NF-κB-related genes commonly In astronauts, PIGR and/or sIgA expression in saliva or blood has induced during both flight and ground infections included the Rel increased following spaceflight48,67,68. However, sIgA levels in protein NFKB2 and the atypical IκB member, NFKBIZ (Supplemen- extraintestinal sites are not always predictive of intestinal tary Tables 4 and 6). RELB and REL, which encode distinct Rel responses69,70. Thus, there is a need for further investigation into proteins which are induced in response to pathogen-associated how spaceflight regulates these intestinal-specific responses. molecular patterns/PAMPs (e.g., flagellin, LPS), were also upregu- iTRAQ analysis further revealed unique differences at the lated in flight and ground infections, respectively (Supplementary protein level in HT-29 cells for flight and ground infections. Tables 4 and 6). In line with these observations, both flight and Keratins 8 and 18 (KRT 8, KRT18), both targets of the Salmonella ground infections led to the upregulation of transcripts encoding effector SipC during invasion56, were downregulated in the several chemokines important for recruiting neutrophils and other infected flight cultures relative to the uninfected flight cultures immune cells to the site of infection, including CXCL1, CXCL2, (Supplementary Table 3). In contrast, these proteins were CXCL3, CXCL8, and CCL20. NFKBIZ encodes NF-κB inhibitor zeta upregulated in response to infection in ground cultures (Supple- (IκBζ), which both represses and activates select NF-κB genes60. mentary Table 3). Infected flight cultures also displayed the IκBζ is rapidly induced in response to infection or exposure to downregulation of two additional cytoskeletal proteins, ACTG1 potent TLR stimuli and is an important inhibitor of nuclear NF-κB and TUBA1C, as well as a retinal dehydrogenase, ALDH1A1. As activity to prevent uncontrolled inflammation61. These findings mentioned above, spaceflight-induced alterations in the unin- confirm that during spaceflight, Salmonella can induce a subset of fected flight cultures in several of these structural proteins relative the known biosignatures of infection in the host at the to the uninfected ground controls, which may indicate the transcriptional level. presence of an altered or dysregulated cytoskeletal landscape at In addition to those commonly observed responses, both flight the starting point for the infection. Spaceflight is known to alter and ground cultures also displayed unique transcriptional the organization and/or expression of structural proteins14,20,21. responses to infection (Fig. 2b, c; Supplementary Tables 4–7). Infection in ground controls induced changes in the expression of DAVID analysis of upregulated genes in the infected flight cultures proteins with various functions, including heat shock/protein indicated that they were enriched for negative regulation of NF-κB folding, RNA-binding, calcium-sensing, and disulfide bond cata- transcription factor activity as well as those involved in lysis (Supplementary Table 3). Heat shock protein HSPA1A was Epstein–Barr virus infection (Fig. 2c). Genes belonging to these upregulated during infection in ground cultures but not in categories overlap with those known to be important for response to infection in flight. This difference between the flight Salmonella infection. Two uniquely upregulated genes of interest and ground infections could possibly be attributed to the fact that belonging to these categories were anti-inflammatory mediator the protein was already 2.4-fold upregulated in flight even in the NFKBIA (IκBα) and NF-κB transcription factor, RELB.IκBα functions absence of infection (see uninfected flight vs. ground comparison

npj Microgravity (2021) 9 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Barrila et al. 5

Fig. 2 Transcriptional responses of HT-29 cells elicited in response to infection with S. Typhimurium during flight or ground culture. a Volcano plots. Differentially expressed genes between infected and uninfected host cells during spaceflight (left panel) or in matching ground controls (right panel). Red: significantly upregulated genes in response to infection. Blue: significantly downregulated genes. Significance determined according to a log2 fold change in expression of at least ±1 and FDR < 0.05. The top 5 significantly up and downregulated genes are labeled. b Venn diagram. Overlapping and distinct genes elicited in response to infection in flight and ground cultures. c, d Uniquely and commonly enriched GO terms and KEGG pathways in response to infection either in flight or ground cultures relative to their respective uninfected controls. Analysis was performed in DAVID 6.8 using upregulated (red) or downregulated (blue) transcripts using an EASE score of 0.05 and Benjamini–Hochberg correction to determine significantly enriched terms and pathways (adjusted P < 0.05). BP biological process, CC cellular component, MF molecular function. Bars in d indicate the results for a flight infection (yellow; flight infected/uninfected) and ground infection (gray; ground infected/uninfected).

Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 9 J. Barrila et al. 6 in Supplementary Table 3). Thus, the flight-cultured cells may have p value = 0.006). In our previous studies, hfq was downregulated had sufficient amounts of HSPA1A protein readily available for in Salmonella in response to both spaceflight and spaceflight response to infection. HSPA1A is a major stress-inducible member analog culture9,12. Deletion of the gene impacted RWV trends in of the HSP70 family and can be upregulated as part of a acid stress resistance and macrophage survival9. Hfq post- cytoprotective response to a variety of stressors, including transcriptionally regulates gene expression by pairing mRNAs infection71. Heat shock proteins have previously shown altered with cognate small non-coding RNAs to either inhibit or facilitate expression (RNA and protein) in a variety of mammalian, plant, translation76. Given the diverse functions of hfq and its ability to and prokaryotic cells during spaceflight or spaceflight analog both positively and negatively regulate translation, its absolute culture, though not in a unified direction72, which is not surprising expression level can change depending on environmental given their diverse functions. For example, multiple HSP70 conditions77. The conditions in our current study (GTSF-2 media, transcripts were upregulated during BRIC-16, an Arabidopsis 37C, 5% CO2) are very different from those in our STS-115 thaliana tissue culture payload aboard STS-131 (the same Shuttle experiment which solely looked at pure cultures of Salmonella mission as the present study)72. (Lennox Broth/LB, ambient temperature). In addition, our STS-115 We also performed a direct comparison between infected flight study was performed in the absence of the host, as all infections and ground cultures under the previously described limitations with microgravity-grown bacteria were done immediately upon (Supplementary Table 8). Direct comparison indicated that landing. In our present study, the bacteria were co-cultured with infection during spaceflight-induced upregulation of host genes host cells that had either been exposed to spaceflight prior to associated with inflammation, response to Salmonella challenge infection or infected as ground controls. Thus, the host cells in our and/or wound healing relative to infected ground controls, current study experienced different environmental conditions including CXCL8 (IL-8), CXCL2, CSF2, IL36B, MAPK13, JUN, FOS, (microgravity vs. unit gravity), which could also potentially KLC2, NCF2, FLNA, RHOG, among others (Supplementary Table 8). contribute to observed bacterial differences. In addition, the IL-8 chemokine expression can be stimulated by both LPS and cellular mRNA and protein concentration of hfq is tightly flagellin73. Elevated IL-8 levels were previously observed in a controlled by autoregulation and mRNA expression is also under postflight challenge using purified LPS from Escherichia coli (E. coli) control of several promoters, including σ32 (encoded by rpoH,a and astronaut immune cells, the latter collected either in-flight or global stress response regulator that was also upregulated in at landing (relative to pre-flight levels)4,65. Interestingly, both response to spaceflight, Supplementary Table 11)77. It is unlikely increased and decreased IL-8 responses have been observed that the Hfq protein was completely suppressed in ground following postflight LPS challenge in crew immune cells74.An cultures given its important role in diverse cellular functions; overall blunted response to E. coli LPS was observed when however, since bacterial proteins were not detected in our endothelial cells were challenged during spaceflight, including proteomics analysis these levels are unknown. If indeed elevated only a modest increase in IL-8 expression by flight cultures relative amounts of Hfq protein were present in the ground cultures to ground75. As is the case in terrestrial studies, IL-8 responses to relative to flight, it is possible that mRNA expression was inhibited LPS challenge in the context of spaceflight will depend on the in the ground cultures via autoregulation. Future studies will seek type of LPS and host cells evaluated, as well as LPS exposure in the to confirm these gene expression trends and identify the context of a live pathogen. In this study, TLR4 was significantly associated regulatory mechanisms. downregulated in the infected flight samples relative to infected To our knowledge, this study was the first to examine how ground cultures (Supplementary Table 8), which suggests that the human cells respond to microbial infection when both the host enhanced expression of pro-inflammatory mediators (e.g., CXCL8) and pathogen are simultaneously exposed to spaceflight. In in the infected flight cultures may rely more heavily on other addition, it is the first global transcriptomic and proteomic interactions, such as flagellin-mediated interactions with TLR5. profiling of human intestinal epithelial cultures during spaceflight Alternatively, the kinetics of infection may have been altered (either infected or uninfected). Although the sample size was small between flight and ground cultures, where TLR4 expression in the in this study due to technical limitations, the results from this infected flight cultures may have occurred outside of our analysis investigation suggest that spaceflight altered the transcriptional time point. Future studies following the in-flight kinetics of and proteomic profiles of both uninfected and infected host cells. infection would provide insight in this regard. iTRAQ analysis Spaceflight culture of the uninfected cells led to the upregulation indicated that infection during spaceflight induced downregula- of genes associated with IFN-I signaling and decreased expression tion of multiple cytoskeletal genes and retinal dehydrogenase 1 of multiple bioenergetic pathways, suggesting the potential for relative to infected ground cultures (Supplementary Table 3). decreased proliferation relative to ground cultures. Decreased Direct comparison of the bacterial transcripts between infected proliferation could be due to the changes in tumorigenic flight and ground cultures indicated 328 upregulated genes and 1 potential, differentiation or DNA damage responses. Salmonella downregulated gene (Supplementary Table 11). Transcripts infection induced a core set of pro-inflammatory responses in associated with metabolism (glycolysis/gluconeogenesis, TCA both conditions, but spaceflight culture also induced unique cycle, purine and pyrimidine metabolism, amino sugar and transcriptional and proteomic responses not observed in the nucleotide sugar metabolism), protein translation, antibiotic identical ground controls. Trends observed between infected synthesis, amino acid biosynthesis, RNA degradation, and flight and ground samples were consistent with a heightened biosynthesis of siderophore group non-ribosomal peptides were response of host cells to infection with Salmonella during upregulated in flight. We also observed the upregulation of the spaceflight, including amplified induction of genes encoding AcrAB-TolC multidrug efflux pump (Supplementary Table 11). pro-inflammatory mediators and wound healing. These findings Though we did not see changes in Salmonella pathogenicity island are important to consider in the context of previously observed 1 or 2 (SPI-1 or SPI-2) virulence genes, transcripts encoding major changes in astronaut intestinal microbiota3,48,78, where some global virulence and/or stress regulators rpoS, rpoE, and rpoH were alterations were linked with decreased abundance of anti- upregulated in flight relative to ground controls. inflammatory species and increases in select pro-inflammatory Another global regular, hfq, was found to be expressed above species3,78. In addition, the use of nutritional countermeasures to background levels in the flight cultures, but not the ground prevent or mitigate infection including the modulation of cultures (Supplementary Tables 9 and 10). However, in the direct nutritional supplements2,10 or probiotic microbes for intestinal comparison between infected flight and ground cultures, while health during spaceflight (including for protection against we did observe an upward trend in hfq expression during pathogens)79–81 are active areas of research and have shown spaceflight, it was not significant by FDR (7.8-fold; FDR = 0.24, promise in ground-based studies. Our investigation adds to the

npj Microgravity (2021) 9 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Barrila et al. 7 growing number of studies aimed at mitigating infectious disease In-flight operations risks to humans during spaceflight missions and for the general At approximately L + 3 h, astronauts activated the on-orbit programming public on Earth. of the CCM. On mission elapsed day 11, the CCM was programmed to automatically inject 1 mL (~1 × 107 bacteria) into a subset of the hollow fiber bioreactors to enable infection at the apical surface of host cells. Six METHODS hours after the bacteria were injected, RNAlater II was injected into the Mammalian cell lines, bacterial strains, and flight hardware designated bioreactors and the rails were turned off to maintain sample temperature at 29–30 °C until landing. The human colonic epithelial cell line, HT-29 (ATCC, HTB-38), was cultured at 37 °C and 5% CO2 in GTSF-2 media (Hyclone) supplemented with 10% heat-inactivated FBS (ThermoFisher Scientific), 2.25 g sodium Postlanding processing bicarbonate (Fisher Scientific) and 5 mL insulin transferrin selenite STL-IMMUNE landed at KSC at 9:08 a.m. on April 20, 2010. All bioreactors (Sigma-Aldrich) per liter. Media used to expand cells as monolayers and bags were removed from the rails and stored at 4 °C. Each reactor was contained Penicillin–Streptomycin (10 units/mL and 10 µg/mL, respec- carefully opened, cells and hollow fibers were maintained in RNAlater II at tively; Sigma-Aldrich) and fungizone (0.5 µg/mL). Flight culture media −80 °C until further processing. Inspection of the remaining volumes in the contained only streptomycin (Sm) and fungizone. Infection studies were bacterial bags post flight indicated that all infected flight cultures received performed with wild-type S.Typhimuriumχ3339, a mouse-passaged the intended 1 mL bacterial injection. Infected ground cultures received 0.8 24 derivative of SL1344 which is naturally Sm-resistant . Bacteria were and 0.4 mL injections for two replicates used for both the RNA-seq/iTRAQ cultured in Lennox Broth (LB) containing 0.3 M NaCl prior to loading in analysis, and the third replicate received a 1.2 mL injection (this sample the hardware, as described below. HT-29 cells were cultured in 2.2 mL used for iTRAQ analysis only). For processing, RNAlater II-fixed samples fi hollow ber bioreactors (Spectrum Labs) that were housed in the Cell were thawed at 4 °C. Samples were concentrated using a Vivaspin 20 Culture Module (CCM; Tissue Genesis). The CCM hardware enabled (0.2 µm) concentrator. Cells were allowed to settle at 4 °C, the supernatant automated media perfusion, addition of bacteria to perform infections was removed and then processed for iTRAQ and/or RNA-seq analysis. and sample fixation. Flight comparisons were made to identical ground controlshousedinthesamehardwareatNASAKennedySpaceCenter (KSC). The flight hardware and experimental layout are shown in Fig. 1. Isobaric tags for relative and absolute quantitation (iTRAQ) analysis S Proteomic analysis using iTRAQ was performed on total cellular protein Preparation of . Typhimurium fl = χ from HT-29 cells from triplicate ight and ground bioreactors (n 3for Ten days before launch, S. Typhimurium strain 3339 was streaked from a flight and ground infected and uninfected cultures). Cells were lysed with 1 glycerol stock onto LB agar plates containing 100 µg/mL Sm and incubated cell bed volume of RLT buffer (Qiagen) supplemented with cOmplete EDTA- at 37 °C overnight. Bacterial colonies were then used to inoculate overnight free protease inhibitor (Roche), vortexed and passed 5–7 times through a cultures grown for 15–16 h. with aeration in LB media containing 0.3 M NaCl 25G needle to facilitate lysis. After a 10-min incubation on ice, lysates were and 100 µg/mL Sm at 37 °C. Cultures were pelleted at 7000 r.p.m. and centrifuged at 12,000 rpm for 10 min at 4 °C. Lysates were added to a resuspended in Dulbecco’s Phosphate Buffered Saline (DPBS; Invitrogen). desalting column (Pierce) and eluted by centrifugation at 1000 × g for Bacteria were quantified by plating for viable CFU per mL and diluted to 2 min. From each condition 25 μg protein sample was prepared according 1×107 CFU/mL in DPBS to maintain the bacteria as viable but not actively to the manufacturer’s instruction (iTRAQ reagents—4plex Application kit, growing. On L-7, flight bacteria bags were filled with 2 mL of the cultures Applied Biosystems). Briefly, after protein reduction, alkylation, tryptic and maintained at room temperature until attachment to the flow paths digestion (Promega), and labeling with iTRAQ 4plex, peptide samples were 2 days prior to launch (L-2). quenched before pooled and vacuum-dried. The combined iTRAQ-reagent labeled sample was resuspended in 2% acetonitrile and 0.1% trifluoroacetic Preparation of hollow fiber bioreactors, media bags and acid before separated with a 145-min gradient elution (using a Tempo LC fixatives MALDI Spotting system (Applied Biosystems)) and analyzed with a 4800 fl Sterile bioreactors were wetted with 70% ethanol (10 mL) and then rinsed MALDI-tandem time-of- ight (TOF/TOF) mass spectrometer (Applied 82 fi fi in triplicate with 10 mL DPBS. A 1 mg/mL collagen I solution (bovine, Biosystems) as described . Protein identi cation and quanti cation was Corning) in DPBS was then introduced through the ECS ports to coat the performed with ProteinPilot 4.0 software using Paragon algorithm. The hollow fibers. After 1 h, bioreactors were rinsed in triplicate with DPBS search parameters were as follows: iTRAQ 4plex (peptide labeled), trypsin as through the ECS ports to remove unbound collagen. Bioreactors were the digestion agent, UniProt database Homo sapiens (created on March rinsed in triplicate with 10 mL of warm flight medium and placed at 37 °C 2009), ID focus: biological modification. Proteins were considered as until ready for loading with HT-29 cells. Flight and ground control bags differentially expressed according to the following criteria: greater than a contained either 60 mL of flight culture media or 40 mL RNAlater II fixative twofold expression change, p value ≤ 0.05 (two-tailed ANOVA). Differential (Ambion) for transcriptomic and proteomic analyses. proteins were identified with at least three unique peptides (>95% confidence, 1% FDR). Identified and quantified proteins by LC MALDI-MS/ MS are shown in Supplementary Dataset 1. Preparation and loading of HT-29 cells into flight hardware HT-29 cells were expanded as monolayers in T-75 flasks. Four days prior to launch, confluent monolayers were washed in warm Hank’sBalanced RNA-Seq Salt Solution without calcium and magnesium (ThermoFisher Scientific), RNA was extracted from flight and ground cultures using remaining dissociated using 0.25% Trypsin-EDTA (Corning), and counted using samples following the proteomics processing. From the remaining trypan blue exclusion to determine cell viability. Approximately 1 × 106 samples, RNA-seq analyses were performed using duplicate flight and HT-29 cells were loaded into twelve 2.2 mL hollow fiber bioreactors ground bioreactors for all conditions (n = 2 biological replicates) except for (Spectrum) through the ECS ports (Fig. 1). Six bioreactors were the infected flight samples, which only had sufficient sample remaining to designated for flight (three infected and three time-matched uninfected evaluate duplicate technical aliquots from a single bioreactor (i.e., n = 1 controls) and six for identical ground controls. Bioreactors were placed at biological; n = 2 technical replicates). RNAlater II-fixed samples were lysed 37 °C and rotated every 15 min until attachment to the CCM rails together with 700 µl Qiazol (Qiagen), vortexed extensively and passed several times with the flight media bags. CCM rails are organizational subunits within through a 25G needle. After a 5-min static incubation at room temperature the CCM that facilitate gas and media delivery. Rails were placed into a with intermittent vortexing, samples were added to MaXtract High-Density 37 °C incubator and monitored for contamination. Media was circulated tubes (Qiagen) together with 100 µl RNase-free water (Qiagen) and 200 µl within the ICS of the fibers during the experiment at a flow rate of 6 mL/ chloroform (Sigma-Aldrich). Samples were then processed using the min. The day prior to launch, the fully loaded rails containing the HT-29 miRNeasy kit (Qiagen) according to the manufacturer’s instructions, cells, media, fixatives and bacteria (maintained in stasis in DPBS) were including on-column DNase-treatment. RNA was quantified using a integrated into the CCM, which was then sealed, powered and the Nanodrop spectrophotometer and the integrity validated using an Agilent temperature set to 37 °C. The CCM was turned over to the KSC integration Bioanalyzer 2100 to confirm an RNA integrity number (RIN) of 7 or greater team at L-18 h. STL-IMMUNE launched aboard Space Shuttle Discovery for all samples. All RIN were between 7 and 8 except for one of the (STS-131) on April 5, 2010 at 6:21 a.m. uninfected ground control samples which had a RIN of 6.4.

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IkappaBzeta is a key transcriptional regulator of IL-36-driven Designed research: C.A.N., J.B., C.M.O., S.F.S., N.H., R.U.H., S.Y., J.P., J.S.; M.M., S.L.C.-W. psoriasis-related gene expression in keratinocytes. Proc. Natl Acad. Sci. USA 115, Performed research: J.B., S.F.S., C.A.N., N.H., N.B., S.D.W., N.Y., K.B., and C.M. Hardware 10088–10093 (2018). design and optimization: C.K., A.Y., C.S., and T.C. Science payload mission manage- 61. Yamazaki, S., Muta, T. & Takeshige, K. A novel IkappaB protein, IkappaB-zeta, ment and support: K.S., N.R., D.L., A.L., R.R.D., R.J.F., C.K., A.Y., C.S., and T.C. Analyzed induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB data: J.B., N.H., S.Y., N.B., C.A.N.; R.U.H. Wrote the paper: J.B. Edited paper: J.B., C.A.N., in the nuclei. J. Biol. Chem. 276, 27657–27662 (2001). C.M.O., S.Y., N.H., K.S., and R.U.H. 62. Chen, X. et al. RelB sustains IkappaBalpha expression during endotoxin tolerance. Clin. Vaccin. Immunol. 16, 104–110 (2009). 63. Le Negrate, G. et al. Salmonella secreted factor L deubiquitinase of Salmonella FUNDING typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and NASA Space Biology NNX09AH40G, 80NSSC18K1478. modulates innate immune responses. J. Immunol. 180, 5045–5056 (2008). 64. Sun, H., Kamanova, J., Lara-Tejero, M. & Galan, J. E. A family of Salmonella type IIi secretion effector proteins selectively targets the NF-kappaB signaling pathway COMPETING INTERESTS to preserve host homeostasis. PLoS Pathog. 12, e1005484 (2016). C.A.N is the Editor-in-Chief for npj Microgravity, C.M.O is Deputy Editor, J.B. is an fl 65. Kaur, I., Simons, E. R., Kapadia, A. S., Ott, C. M. & Pierson, D. L. Effect of space ight Associate Editor and K.S. is on the Editorial board. on ability of monocytes to respond to endotoxins of gram-negative bacteria. Clin. Vaccin. Immunol. 15, 1523–1528 (2008). 66. Jin, M. et al. Responses of intestinal mucosal barrier functions of rats to simulated ADDITIONAL INFORMATION weightlessness. Front. Physiol. 9, 729 (2018). 67. Mednieks, M. H. A. In Oral Tissue Responses to Travel in Space. Beyond LEO - Human Supplementary information The online version contains supplementary material Health Issues for Deep Space Exploration (ed Reynolds, R. J.) (IntechOpen, 2019). available at https://doi.org/10.1038/s41526-021-00136-w. 68. Spielmann, G. et al. B cell homeostasis is maintained during long-duration spaceflight. J. Appl. Physiol. 126, 469–476 (2018). Correspondence and requests for materials should be addressed to C.A.N.

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npj Microgravity (2021) 9 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA