The ‘chemical Darwinian’ definition of life is a ‘self-sustained chemical system capable of undergoing Darwinian evolution’. The true meaning of life, however, is much more complex than this and does not hold true for forms of life that lack Darwinian evolution such as sterile creatures [1]. Existence of life on Earth can be segregated into four major biological assemblies: archaea, bacteria, viruses, and eukaryotes. The co-existence of eukaryotes and bacteria in a symbiotic relationship has a long-chronicled history [2], [3]. The mutualistic nature of this relationship is believed to have left evolutionary footprints in eukaryotic hosts [4]. In myriad flora and fauna populaces, epithelial barrier tissues are inhabited by a plethora of microbial species, that play a critical role in morphological development and epithelial barrier function [5]. Although human microbial interactions have been studied for well over 400 years, studies over the past 50 years have unraveled the critical role of human microbiota in health and disease [6], [7]. It has been established that eukaryotic hosts exist in symbiosis with the commensal resident microbes at different barrier sites.

The initial attempts at profiling microbial communities have been ongoing since as early as 1670’s when Antonie van Leeuwenhoek observed bacteria in human dental plaques. However, it was not until late in the 19th century that attempts were made towards systematically exploring the spatial distribution of microbiome in human tissues.

A couple of centuries later, Joseph Leidy published ‘A Flora and Fauna within Living Animals’ in 1853 which is considered as the holy grail of microbial research [8]. Following this, groundbreaking discovery by Louis Pasteur in developing the germ theory of disease broadened the horizons of understanding the role of microbiome in human health and diseases. He also hypothesized that non-pathogenic microbes are a key player in maintaining homoeostasis in human physiology [9]. Next, the works of Metchnikoff and Escherich highlighted the significance of interaction of the endogenous species with the host organism and how it shapes immunity [10], [11]. In 1890, Robert Koch postulated four criterion that established microbes as a causative agent of diseases in host. Such findings laid the foundation of similar work in the twentieth century that focused on microbes as etiological agents of disease [12]. Until this point in microbial research, only microbes that could exist and proliferate in the presence of oxygen had been studied, hence the studies were performed with an unconscious bias towards disease causing pathogens only. Anaerobic microbes as the endogenous gut microbiota composition started gaining limelight in microbial research after Alfred Nissle isolated Esherichia coli in 1917. He noticed that during World War I, one out of many soldiers did not get affected by dysentery and hypothesized that the protective microbiota in his gut provided him protection. He then isolated the strain and proved the ‘theory of colonization resistance’ where host-associated microbes prevent proliferation of pathogenic microbes in the same niche [13]. Despite such findings the true potential of the field of human microbiota research unveiled in the 1940 s with the advent of methods for anaerobic culturing. It is at this point of time that researchers across the world expressed interests in understanding how the composition of microbial communities residing on our barrier tissues change in healthy vs diseased conditions. With the ability to grow microorganisms in the lab, came its own limitations that included vast quantitative anomalies in the number of cells existing on eukaryotic organisms vs that could be grown in the lab - ‘the great plate count anomaly’. Such observations expanded the horizons of microbiota research that opened the doors for sequencing based approaches to study microbes that could not be grown in the lab. Yet another cornerstone in bringing microbial research to the forefront was fecal microbiota transplant (FMT). Eiseman et al., in 1958 published that FMT could successfully cure patients suffering from pseudomembranous colitis [14]. With the popularization of human microbiota research, and the advent of sequencing-based methods, there has been enormous findings characterizing the true potential of microbial-host interactions and the role they play in health and diseases. However, the key to understanding the role of microbes on human health is to study them at the interface of interaction with cells of the eukaryotic system. To truly uncover the underlying mechanism of microbial-host interaction, they have to be captured in action as a snapshot in time. Fig. 1 briefly summarizes the hypotheses and pioneering discoveries in the field of microbial research that paved way to understand the mechanism of host-microbiota symbiosis.

Mammalian barrier epithelial tissues like gut, lung, skin, etc. are exposed to the external environment which makes them ecological niches to house tissue resident microbiota of the organism [15], [16], [17]. Hence, it is of mutual interest to both, the host, and the microbiome to preserve the barrier integrity leading to a symbiotic relationship between the two [18]. Whereby the host epithelial tissue provides habitat and nutrients to the resident microbial populations and in return, the microbiome provides nutrients like vitamin B12, fiber digestion, etc [19]. In addition, recent studies have indicated that the human commensal microbiome plays an active role in educating and training the host immune system to recognize commensal microbial species and react against invasive and pathogenic microbes [20]. The eukaryotic immune system in turn works to maintain functionality in a way that sustains the barrier tissues to respond to microbiome exposure. In this manner, exposure to the microbiome at the barrier tissues primes the mammalian host immune system [21]. An extensive profiling of host-microbiome interactions in the milieu of epithelial microbial niches is crucial to our understanding of the microbial influence on host immunity. To gain an understanding of the crosstalk, a reasonable approach would be to investigate the first interactions of the microbiota with the immune cells of the human body.

For the longest time in human microbiota research, microbes were considered as an entity that function against the host immune system. Thus, most studies were performed with focus on host defense against pathogens [22]. However, with the discovery of the plethora of commensal microbes residing in the gut, the concept of eukaryotic organisms being an holobiont gained more attention. Scientists began to understand that eukaryotic organisms exist in symbiosis with the commensal population residing at the nexus of epithelial tissues [23]. Hence, further discoveries in this field demanded that the focus be shifted from cataloguing host cell types and microbiome population in isolation to studying their interactions. To gain further understanding of this, an example could be gut, the most intricate barrier tissue in the human physiology. A brief conceptual idea of how host-microbiome interactions look like in action can be seen in Fig. 2. However, a prevailing lacuna in the field has been lack of techniques that allow us to look at both microbes as well as eukaryotic cells in one single assay.

Several studies during the last few decades have highlighted the importance of the microbiome in human health and diseases [24]. Starting from immunologically conditioning the foetus to contributing to heterogeneity in the tumor microenvironment, microbial communities play an active role throughout human life [25], [26], [27], [28]. Hence, it is of utmost significance to investigate and characterize the crosstalk between the microbial community and the immune cells. There have been in-depth studies and investigations on the microbial community and their functional characterization as well as detailed research on their contribution to the host immunological ecosystem [29], [30]. However, to study the influence of the microbiome on the host immune cells, they have to be studied together in order to obtain a snapshot of the microbiome interacting with the immune cells in action [31]. Certain microbial species co-localize with distinct immune cell types of the host and interact to form a tight nexus [32]. Each of these niches harbors unique antigenic and metabolic characteristics that enable them to maintain barrier integrity and homeostasis [33]. In cases of barrier breaches, such localized niches are disrupted [34]. Thus, it leads to inflammatory immune responses triggered by a breach in barrier. Hence, a comprehensive understanding of the immune landscape across discrete niches in different regions of the barrier tissues is crucial to investigate the underlying mechanism of how the microbiome contributes to disease progression and pathogenesis [35]. The functioning of several physiological properties in eukaryotes can be attributed to the spatial organization of the cells within the system. The past few years have witnessed an exponential rise in methods that leverage spatial gene expression to recognize genes in a spatiotemporal context [36], [37], [38], [39], [40]. ‘Spatial transcriptomics’ can be elucidated as the method of spatially localized quantification of the expressions of mRNA within a tissue [39], [41], [42]. However, given the immense impact of the microbiome in human health and disease, there is a dearth of spatial techniques that allow mapping of microbial presence within a tissue. Recent studies on the microbiome in the context of human physiology have highlighted that essential aspects of metabolism and immunity are distinctly linked to the microbiome [25], [43], [44], [45], [46]. The gut microbiota plays an active role in disease progression in humans [28]. For example, in conditions such as ulcerative colitis, diabetes, obesity, and even colon cancer, the gut microbiota can be attributed as one of the contributing factors [47]. However, there has been a dearth of scientific evidence on how the host-microbiota dialogue differs in dysbiosis and disease. Moreover, it has been observed that the spatial organization of the gut microbiome is crucial to maintaining homeostasis in human physiology [48]. Disruption of these well-defined microbiome networks often leads to pathogenesis and disease progression [49], [50]. Hence, there needs to be a shift in focus from just cataloging the bacterial diversity in isolation to investigating the spatial architecture of the microbiome in the context of host-microbiome dialogues. Recent spatial studies have demonstrated interesting findings such as the microbiome community geographically closest to the host inner mucus layer having a distinct spatial organization that is composed of a richer microbial community as compared to the other tissues [35], [51]. Such unique spatial features are thus attributed to having an active role in maintaining the stability of the barrier tissue [52]. Thus, the spatial co-localization of the host-microbiome entities is functionally significant to maintaining homeostasis [53]. The success of a microbe in colonizing the host to establish a commensal relationship and even pathogenesis can be attributed to the integrity of the host-microbiome spatial networks [54], [55]. Hence, in-depth characterization of these spatial niches are of relevance to understanding the underlying cause of pathogenesis and disease progression [56]. Given the importance of spatial organization of the microbiome in host immunity, in the current scientific paradigm, there are limited applications that aim for simultaneous profiling of the host and microbiome in a spatiotemporal manner. There has been significant progress in transcriptomic profiling of eukaryotic host genomes including advances in single-cell genomics that are curated to investigate the cellular composition of tissues or certain resident microbes [57], [58], [59], [60], [61]. However, in these cases essential spatial information is lost during the experimental procedure. Additionally, for microbial profiling, certain metagenomic sampling techniques or spatial imaging techniques have come to light that seems promising [62], [63], [64], [65]. Most of these techniques are still limited by the region of interest and level of taxonomic classification in identifying the microbiome [48]. To profile the spatial niches that capture transcriptomic information from both host and microbiome populations, we need to be able to map their crosstalk spatially [66]. Thus, there exists a need to develop a spatial transcriptomic method that will allow us to map the cell states in situ for simultaneous host-microbiome capture.

The microbiome plays an active role in the initiation, activation, and maintenance of the mammalian immune architecture [45], [67]. The evolution of the immune system takes place with an extensive population of commensal species residing on barrier tissue sites that are termed as the microbiome [35]. Eukaryotic hosts have co-evolved with their commensal microbiome as an ecosystem existing at discrete barrier site niches harboring antigenic and metabolic diversity [68]. The antigenic signals and the metabolites produced by the microbiome engages in constant crosstalk with the host immune repertoire thus leading to commensal-specific immune signaling in homeostasis [44]. Mishra et al. in 2021 reported the underlying mechanism of how this network is established during the early developmental stages of a foetus [25]. The immune-microbiome network established during the gestational period has lifelong implications on the health and immunity of humans [69]. There is a fine-tuning between the microbial presence and the immune system of the body that enables it to identify commensal vs pathogenic signals and prepare the immune system to behave differently in each scenario [20], [70]. Various studies have outlined the role of mucosal barrier sites as a hotspot for host-microbiome interactions [48]. However, these sites are also the ones subjected to maximal environmental damage [46]. When such barrier tissues lose their ability to replenish after enduring multiple environmental insults, it may lead to compromised barrier integrity [71]. Breaking down of barrier integrity can disrupt the spatial architecture and lead to tissue-resident microbes at barrier sites becoming pro-inflammatory and even members of the tumor microenvironment by eliciting immunosuppressive pathways [72]. To gain an in-depth understanding of how the microbiome talks to the immune cells of the eukaryotic host, we need to capture their interaction as a snapshot in time. As the immune-microbiome crosstalk is highly specific, they are co-localized in niches along the epithelial boundary [35]. Hence, such interactions have to be studied in situ such that the intact spatial architecture of these networks are maintained. Due to limitations in scientific technology that allows simultaneous spatial profiling of microbial and host immune cells, they have not extensively been studied together in the past.

Few breakthrough studies in the past year have reported novel techniques to simultaneously profile host transcriptome along with the microbiome. Study by Galeano Nino et al., 2022 reported a novel technique to profile cells of the eukaryotic tramscriptome alongside the tissue resident microbiome in one single assay [73]. Additionally, similar studies have reported other techniques such as ‘spatial meta-transcriptomics’ and ‘spatial host-microbiome’ sequencing to identify microbial-immune niches at the nexus of epithelial tissues. Such studies could shape the future dimensions of understanding microbiome in human diseases [74], [75].