Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

doi:10.21203/rs.3.rs-4183349/v1

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Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

https://doi.org/10.21203/rs.3.rs-4183349/v1

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The continuously remodeled extracellular matrix (ECM) plays a pivotal role in gastrointestinal health and disease, yet its precise functions remain elusive. In this study, we employed laser capture microdissection combined with low-input proteomics to investigate ECM remodeling during Salmonella-driven inflammation. We also probed the mechanosensitive state of fibronectin, a crucial ECM component with diverse functions dependent on its tensile state. While fibronectin fibers in healthy intestinal tissue are typically stretched, we demonstrated their relaxation only hours after infection in intestinal smooth muscles, despite the absence of bacteria in that area. In contrast, within the mucosa, where Salmonella is present starting 12 hours post-infection, fibronectin fiber relaxation occured exclusively during late-stage infection at 72 hours, and was localized to neutrophil clusters. Using N-terminomics, we identified three new cleavage sites in fibronectin in the inflamed cecum. Our work suggests that the ECM remodeling responses of different tissue layers in the intestine are distinct and carefully orchestrated.

In recent years, the prevalence of inflammatory gastrointestinal diseases has exhibited a concerning upward trend¹. An understudied aspect of the underlying mechanisms of such diseases is the extracellular matrix (ECM) within the intestinal environment, which offers considerable potential for novel interventions. The ECM is a complex network of proteins and proteoglycans that plays a pivotal role in tissue integrity, maintenance, remodeling, and repair^2–4. In the context of inflammatory gastrointestinal diseases, the ECM is integral to both tissue destruction and repair processes^5,6.

The intestine is a remarkably complex organ, encompassing multiple sub-tissue layers. These layers play vital roles in maintaining intestinal barrier integrity, facilitating nutrient absorption, regulating the immune system, and orchestrating digestion⁷. The intestinal mucosa serves as the primary interface for interactions with luminal content and forms a barrier against pathogens⁸. Meanwhile, the muscularis externa, a smooth muscle layer surrounding the intestinal tube, is responsible for gut motility⁹. Within this intricate system, the ECM provides structural support, elasticity, compressive and tensile strength. Simultaneously, the ECM contains binding sites for signaling factors regulating tissue homeostasis, growth, and repair^10–12. Interestingly, it is not only the biochemical composition but also the physical state of the ECM that is affecting its function¹³. Known ECM remodeling processes are coordinated in the matrix during inflammation¹⁴ and as recently reported also preceding intestinal inflammation¹⁵, further emphasizing the need for molecular examination of the ECM's role in gastrointestinal health.

Fibronectin, an essential ECM protein, facilitates cell adhesion, migration, and differentiation as well as tissue growth and wound healing^16,17. Fibronectin knock-out mice are not viable beyond embryonic day 8, making functional studies challenging¹⁸. Fibronectin is mechanosensitive, showing altered functions depending on the fiber’s tensile state¹⁹. Fibronectin fiber stretching thus can activate cryptic binding sites, inaccessible in the mechanically relaxed protein, which are required for fibronectin fibrillogenesis during fibronectin matrix assembly²⁰. Additionally, interactions with integrins^21,22, collagens²³, interleukin-7²⁴ and tissue transglutaminase 2²⁵, were all reported to be dependent on the tensile state of fibronectin. Despite its functional implications investigated in vitro, the tensional state of fibronectin fibers is mostly unresolved in ex vivo or in vivo settings, primarily due to the lack of appropriate tools. We have previously developed^26,27 and validated a fibronectin fiber tension probe FnBPA5^28–30. This peptide probe is derived from the bacterial fibronectin adhesins of Staphylococcus aureus and presents a powerful tool to measure fibronectin fiber tension in ex vivo cryosections³⁰. FnBPA5 binds with nanomolar affinity, via a multivalent network of backbone hydrogen bonds, to the N-terminal FnI_2 − 5 domains of fibronectin exclusively in a low tensional state. In stretched fibronectin fibers the distance between these four FnI domains is increased, thereby destroying the multivalent binding motif such that the FnBPA5 peptide cannot bind any longer with high affinity³⁰. Notably, the FnBPA5 probe cannot distinguish between fibers that are structurally relaxed due to enzymatic cleavage, or due to the presence of other load-bearing ECM elements, such as more rigid collagen fiber bundles²³.

Here we investigate ECM biology in the oral streptomycin mouse model of Salmonella enterica subspecies enterica serova Typhimurium (S. Tm) infection. This is a highly reproducible model with a well-resolved and reproducible timeline of inflammation^31,32, that allows mapping of new findings to defined steps in the inflammatory response against the invading bacteria³³. A plethora of host defense mechanisms including secretion of antimicrobial peptides, expulsion of infected epithelial cells, recruitment of neutrophils and other inflammatory cells, are involved in infection control^32,34–36. This limits the systemic dissemination of S. Tm. However, these defense mechanisms also drive extensive tissue damage in the intestine as well as collateral damage to the commensal microbiota^37,38.

In this study, we employed laser capture microdissection coupled to data independent acquisition (DIA) tandem mass spectrometry (LC-MS/MS) to investigate tissue-layer specific changes in ECM in inflammation. Our analysis revealed both similarities and differences in the host's response across the individual tissue layers. Importantly, we present for the first time evidence of a spatial correlation between the relaxation of fibronectin fibers, which succeeds the infiltration of neutrophils in the inflamed intestine. These findings shed new light on the intricate interplay between mechanical forces, ECM remodeling and immune response altering protein expression, as well as fibronectin fiber tensional state in a highly defined manner. Finally, we have measured tissue-layer-specific proteomes for both the healthy and Salmonella-infected mouse cecum, and present these datasets as a resource to the field.

Spatial proteomics analysis of healthy and severely inflamed mouse ceca

To elucidate the tissue layer specific proteome of healthy and severely inflamed mouse cecum, we employed a laser capture microdissection (LCM) approach coupled to high sensitivity LC-MS/MS-based proteomics. The LCM technique facilitated targeted isolation of discrete regions within the cecal tissue, offering an unprecedented level of spatial resolution. Infection with 5x10⁷ CFU of S. Tm by voluntary feeding, resulted in high intestinal (10⁹ CFU / g of cecum content) as well as systemic counts (~ 10⁵ CFU / g of spleen) of S. Tm bacteria from day 1 post infection (Fig. S1)^31,39. Mucosa and muscularis externa tissue areas were selectively collected for both healthy and inflamed (day 3 post infection (p.i.)) conditions using LCM as depicted in Fig. 1a. In total we detected 7668 proteins and found that 57% of those were ubiquitously found in all the four biological conditions (healthy mucosa, healthy muscularis externa, inflamed mucosa and inflamed muscularis externa), while we identified more proteins specific for the mucosa compared to the muscularis externa independent of the health status (Fig. 1c).

Functional analysis of proteins detected solely in the inflamed conditions

In total 56 proteins were detected only in the inflamed mucosa, while124 proteins were found only in the inflamed muscularis externa (based on at least three biological replicates). Functional analysis using Metascape⁴⁰ confirmed the involvement of these proteins in the immune response against bacterial infection (Fig. 1d). Interestingly, many highly significant gene ontology (GO) terms involved in immune response presented an increased significance in the muscularis externa or were even found only in this sub-tissue layer (GO: 0002252 - immune effector process and GO: 0035456 - response to interferon-beta, respectively). Identification of lipopolysaccharide signaling (GO: 0031664 - regulation of lipopolysaccharide-mediated signaling pathway) specifically in the mucosa reflects the location of invasive bacteria at this timepoint^35,41. Remarkably, we detected laminin subunit gamma 2 (Lamc2) only in the inflamed mucosa, suggesting that the epithelial basement membrane gets partially degraded, whereas it remains below the detection limit in the healthy mucosa. Laminins are important structural components of the epithelial basement membrane⁴². Lamc2 has been associated with epithelial cell migration following cleavage by MMP2 in wound scenarios⁴³, and was reported to be upregulated in dextran sulfate sodium induced intestinal inflammation⁴⁴. Investigating the implications of upregulated Lamc2 in the inflamed intestinal mucosa could provide valuable insights into tissue responses and might even be used for therapeutic applications in the future. We also detected interesting protease candidates likely involved in ECM remodeling. In mucosa and muscularis externa, we could detect matrix metalloproteinase 3 (MMP3), a well-known matrix remodeling metalloprotease⁴⁵, and haptoglobin, which has antimicrobial and antioxidant activity and is involved in hemoglobin clearage and immune response^46,47. Specifically for the inflamed muscularis externa, we identified Cathepsin G, known for its ECM remodeling functions and potential fibronectin cleavage capability⁴⁸, and granzyme A, a serine protease with recently demonstrated ECM remodeling function⁴⁹ (Fig. 1b).

Protein abundances change in a tissue layer-specific manner upon S. Tm-induced inflammation

In addition to the proteins exclusively detected in the inflamed conditions, we found a plethora of proteins exhibiting differential expression between the inflamed and healthy states. Predominantly upregulated proteins within the mucosa were associated with the inflammatory response (e.g. Sting1, Igtp, and Zbp1), including functions such as cytokine production, neutrophil degranulation, and MAPK family signaling (Fig. 2a). Notably, a substantial downregulation of metabolism-related proteins (e.g. Ass1, Dld, and Auh) in the mucosa (R-MMU-71291 - Metabolism of amino acids and derivatives) was evident, underscoring a metabolic shift associated with inflammation. Building on these observations, our focused analysis within the mucosa revealed 33 proteins experiencing significant upregulation, while 48 proteins underwent significant downregulation in the inflamed state compared to the healthy tissue layer (Fig. 2c). In the mucosa, neutrophilic granule protein and the lymphocyte antigen 6 family member A (Ly6a) are specifically upregulated (Fig. 2c + e), reflecting the strong infiltration of neutrophils into this tissue area. Additionally, expression of the leukocyte-specific integrin subunits β2 and αM is increased (Fig. 2e), likely reflecting the invasion of immune cells^50,51. Several of the detected proteins are also upregulated in other inflammatory intestinal diseases: extracellular matrix protein 1 (ECM1), which was reported to be elevated in inflammatory bowel disease (IBD)⁵² and tubulointerstitial nephritis antigen like 1 (Tinagl1), a matricellular protein with protease activity upregulated in gastric cancer and involved in MMP regulation⁵³, as well as in Heliobacter pylori induced gastritis⁵⁴. Together with Tinagl1, other proteins with proteolytic activity such as plasminogen (Plg), furin and proteasome subunit beta type 8 (Psmb8) were also upregulated (Fig. 2b). As a representative instance of downregulated metabolism in the inflamed mucosa (Fig. 2e), the decreased expression of cytochrome P450 C55 is noteworthy, as this underscores the broader metabolic shift within the inflamed setting.

In the muscularis externa, we observed no significant upregulation of leukocyte-specific integrins. Instead, functional analysis unveiled the downregulation of fatty acid metabolism upon inflammation (R-MMU-8978868 – Fatty acid metabolism) and the upregulation of some similar inflammatory response pathways as observed in the mucosa. Specifically, we detected 57 significantly upregulated and 9 downregulated proteins in the inflamed condition compared to the healthy one (Fig. 2d). Upregulated proteins with proteolytic function such as glutathione hydrolase 5 proenzyme (Ggt5), caspase 12 or Psmb10 were detected only in the muscularis externa (Fig. 2b). One interesting protein upregulated in this sub-tissue layer was Bst2, also known as tetherin or CD317 (Fig. 2f), which is a lipid raft associated protein with potential ECM-cell interaction function⁵⁵ and which is expressed in response to interferons. Muscularis externa specific downregulation was observed for the matricellular glycoprotein Secreted Protein Acidic and Rich in Cysteine (SPARC) (Fig. 2f). SPARC is known for its ability to promote collagen fibril assembly, by stabilizing microtubule networks via integrin-linked kinase binding to integrins^56,57, and its upregulated expression was shown in aging mice to induce the inflammatory interferon-response in macrophages, promoting their conversion from anti- to pro-inflammatory⁵⁸. Reports on SPARC regulation in intestinal disorders are still limited with a recent study showing increased colonic mucosal mRNA levels in active ulcerative colitis⁵⁹. This interesting discrepancy between chronic and severe acute intestinal inflammation and tissue sub-layers (as well as human vs. mouse) is intriguing but its functional understanding goes beyond the scope of this study. Additionally, we detected proteins that were differentially regulated in both tissue levels (mucosa and muscularis externa). Fibrinogen beta chain (Fgb) was detected to be upregulated (Fig. 2c, d, f) showcasing the importance of the blood clotting system. Selenium-binding protein 1 (Selenbp1, also known as methanethiol oxidase) was downregulated upon severe inflammation (Fig. 2e + f). Selenbp1 is found in mature enterocytes catalyzing the conversion of methanethiol to hydrogen sulfide and other components. It was also reported to be downregulated in various cancers, associated with poor prognosis^60,61.

The distribution of identified matrisome proteins is consistent across both sub-tissue layers and disease states

We noticed variations in the expression of integrins and ECM remodeling proteins, including MMP3, Cathepsin G, and members of the serpin family. This sparked our interest in understanding the distinctions within the matrisome related proteins, across the four groups. Intriguingly, our analysis revealed that the percentage of detected matrisome components remained remarkably consistent across all four conditions (3–5%) (Fig. S2a). Furthermore, the distribution of the individual subcategories (proteoglycans, glycoproteins, collagens, ECM remodeling factors, ECM-affiliated proteins, and secreted factors) exhibited no significant changes between the sub-tissue layers and the physiological state (Fig. S2b). Given that ECM remodeling can be more subtle than changes in these overarching categories and considering our identification of elevated MMP3 levels - a well-known protease capable of cleaving the ECM component fibronectin - we directed our focus towards exploring potential alterations in fibronectin fibers within the two distinct sub-tissue layers.

Probing healthy cecal tissue using a fibronectin fiber tension probe

Fibronectin, known for its broad functions regulating tissue growth, remodeling and repair processes¹¹ and the mechano-sensitivity of its binding sites, partially activated or destroyed by fiber stretching²⁰, is an interesting candidate to compare ECM remodeling in healthy as well as inflamed intestines. Additionally, previous research revealed that fibronectin fibers are highly tensed in other healthy organs, while their tension is progressively lost in tumor stroma^28,29, motivated us to probe the conformational state of fibronectin fibers in the healthy as well as the inflamed intestine. To study the tensional state of fibronectin fibers, we applied the bacterially derived FnBPA5 peptide, which identifies relaxed fibronectin fibers via binding of the N-terminal fibronectin domains FnI_2 − 5 in a mechanosensitive manner^26,27. To control for non-specific peptide binding we applied a scrambled peptide version with a shuffled amino acid sequence³⁰. Fibronectin and FnBPA5 co-staining showed abundant fibronectin signal across all tissue layers, but no presence of low tensional state fibronectin fibers in healthy ceca (Fig. 3a + b, top row). This observation is in line with previous observations in other healthy organs showing no FnBPA5 signal²⁸. We recently reported similar results in the context of intestinal edema, where fibronectin fiber degradation and thus relaxation seems to be inhibited by protease inhibitors⁶².

Fibronectin fiber tension in the smooth muscle layers strongly decreases early during S. Tm infection

At early timepoints, we only observed FnBPA5 tensional state probe-binding in the muscularis layers: the muscularis mucosa (located between the mucosa and the submucosa) and the muscularis externa. FnBPA5 staining becomes prominently visible in mice 24 h p.i. as illustrated in Fig. 3. However, even at the 6 h p.i. timepoint, a discernible increase in signal intensity can be quantified through pixel-by-pixel analysis (Fig. 4c). During the time course of the Salmonella infection, the muscularis signal increases dramatically and results in 80% of FnBPA5-positive pixels on day 3. Visualizing the distribution of the FnBPA5 pixel intensities clearly shows few high-intensity pixels on day 1, shifting to broadly increased fluorescence intensity on days 2 and 3 (Fig. 4d). This may represent fibronectin relaxation due to the overall muscle contraction induced by the inflammatory response. The cecum shrinkage and its morphological implications were recently described by us in detail⁶³. Of note, we controlled for post-mortem muscle changes as 1) no FnBPA5 signal is observed in the healthy intestinal muscularis and 2) ceca were manually filled with mounting medium prior to embedding to avoid non-physiological tissue collapse.

Mucosal fibronectin fiber tension decreases in spatially defined areas and only in the late stage during the S. Tm infection

Focusing on the mucosal area of the tissue, the FnBPA5 signal and thus fibronectin fiber tension changes are only detectable at late timepoints. First on day 2 p.i. sporadic distinct small pixel clusters of FnBPA5 appear. On day 3 the FnBPA5 signal becomes prominent forming larger clusters in the mucosa (Fig. 3b, bottom row). The average FnBPA5-positive pixel coverage in the mucosa stays low in comparison to the muscularis externa signal. On day 2 p.i., we observed a slight increase and on day 3 the FnBPA5-positive mucosal tissue area accounts for 5% of the total mucosal area (Fig. 4a). As can be seen in the intensity histograms, the FnBPA5 pixel intensity shows higher values on day 2 and day 3 compared to the control and the early timepoints of the infection.

In tumor sections FnBPA5 was reported to be in spatial proximity with collagen fiber bundles as visualized by second harmonic generation (SHG)²⁸. Additionally, it is known that during wound healing processes in the skin, collagen III and in later stages collagen I are secreted to support healing⁶⁴. The measured SHG signal at day 3 in the mucosa turned out to be quite low, in contrast to cancer stroma, and we did not observe any elevated SHG signal in the locally elevated mucosal FnBPA5 regions (Fig. S3). This indicates that the fibronectin fibers relaxation in the mucosa is not caused by thick collagen fibers taking over tissue strain²³.

Spatial correlation of relaxed fibronectin fibers and infiltrating neutrophils in the mucosa on day 3 p.i.

Neutrophil invasion into the mucosa as well as their swarming behavior⁶⁵ are well-known phenomena⁶⁶ and our proteomics data corroborated this by exhibiting enhanced leukocyte infiltration via the presence of neutrophilic granule protein and Ly6a. Thus, we contemplated the possible involvement of neutrophils in the fibronectin fiber relaxation process in the inflamed mucosa. Neutrophils are not only known as the first line of defense against bacterial infections, but also secrete a lot of proteases upon activation, many of which are known to be able to cleave fibronectin⁶⁷. Staining with an anti-Ly6B.2 antibody showed appearance of first clusters of neutrophils in the mucosa after 24 h p.i. (Fig. 5a). On day 2 and 3 the number of neutrophils increased, with an increasing number of clusters being progressively observed. The number of Ly6B2-positive cells increased to 25% of the total mucosal cells on day 2 (Fig. 5b, Fig. S4). Quantification of FnBPA5 signal in Ly6B.2-positive cluster regions versus Ly6B.2-negative regions in the mucosa on day 3 showed a significant (p = 0.003) increase of FnBPA5 in the areas of Ly6B.2-positive clusters. Surprisingly, this phenomenon only appears on day 3, not on day 2 of the infection. Thus, the mucosal FnBPA5 signal is spatially strongly correlated with neutrophil clusters in the late stage of the infection. Since the area fraction of these regions is small in comparison to the whole mucosal area, the localized FnBPA5 signal does not significantly affect the averaged mucosal FnBPA5 tissue coverage. When solely considering the neutrophil cluster areas, the quantified FnBPA5-positive tissue area increased from 5% to almost 20% (Fig. 5c). This indicates that matured neutrophil clusters, observed in the cecal mucosa of S. Tm infected mice, are closely associated with fibronectin fiber relaxation either directly or indirectly.

Terminal amine isotopic labeling of substrates identifies 3 sofar unknown cleavage sites in fibronectin type III domains elevated in the inflamed cecum

To further focus on and analyze proteolytic cleavage events in healthy versus severely inflamed cecum tissue, we utilized the terminal amine isotopic labeling of substrates (TAILS) workflow. With this method it is possible to detect both natural protein N-termini and protease generated neo-N-termini by labeling protein N-termini prior to the depletion of internal tryptic peptides^68,69. While analysis of LCM derived tissue areas would have provided more insights into fibronectin cleavages, we could not provide high enough protein levels with this method. Instead of the LCM tissue pre-processing step, we thus used whole cecum tissue (including all tissue layers) for the TAILS analysis and included two healthy mice and two severely inflamed mice (day 3 S. Tm infection) in technical replicates. Following LC-MS/MS analysis, the data processing was performed using CLIPPER 2.0, a novel tool integrating annotation, statistics and visualization of degradomics datasets⁷⁰. In total, we identified 3407 proteins and 2939 N-termini from the cecal samples. 827 of these N-termini were acetylated, while 2112 were non-acetylated (Fig. S5). We detected 101 unique N-termini that were significantly elevated in the inflamed cecal tissue. Amongst those were interesting peptides from S100-A9, complement C3 and haptoglobin. The haptoglobin cleavage site at (R(102).(103)IIGGSM) is located right in between the haptoglobin alpha and beta chain, hinting at induced activation of its important functions ranging from antibacterial activity to hemoglobin capturing. In the healthy cecal tissue we detected 162 upregulated N-termini with peptides in cathepsin B and meprin A subunit beta, indicating towards higher activation in the healthy state. In the context of this work, we further focused on fibronectin cleavage to investigate if there might be significant cleavage events in the inflamed conditions, which could possibly explain the increase in FnBPA5 signal. Indeed, we found 3 new peptides to be significantly higher represented in the inflamed cecum compared to the healthy one. Two of them are located in fibronectin type III domain 6 (sequence positions Thr¹¹⁴⁹ - Arg¹¹⁵⁶ and Thr¹¹⁵¹ - Arg¹¹⁵⁶) and one is located in fibronectin type III domain 17 (sequence position: Asn²²⁵⁹ - Arg²²⁶⁹) (Fig. 6). All 3 N-terminal cleavage sites (P1 position at Tyr¹¹⁴⁸, Tyr¹¹⁵⁰ and Tyr²²⁵⁸, respectively) have not been reported before and cannot be mapped to specific proteases. Interestingly though, all three cleavage sites have tyrosine in their P1 position, which indicates potential chymotryptic-like protease activity. Follow-up experiments will be necessary to understand which specific proteases are responsible for these fragments and whether these peptides have specific functions in the severely inflamed cecum.

The need for enhanced understanding and improved treatment options for gastrointestinal diseases is of central relevance. Investigating ECM alterations during intestinal inflammatory processes provides a novel and promising avenue for addressing this necessity. Here, we combine a well-studied intestinal inflammation model, mechano-sensitive imaging, and LCM-coupled MS-based proteomics. With this approach, we identify spatiotemporally definite ECM and proteome changes occurring within the two sub-tissue layers mucosa and muscularis externa during S. Tm induced intestinal inflammation. Of equally important significance, we discover and describe three new cleavage sites in fibronectin using degradomics. Though a protein specificity motif search did not allow for the identification of candidate proteases, this protease must be upregulated in the inflamed cecum.

While both sub-tissue layers exhibit a common set of differentially expressed proteins indicative of a general inflammatory response against the S. Tm infection, distinct characteristics (e.g. regulation of lipopolysaccharide-mediated signaling pathway for the mucosa vs. response to type II interferon specific for muscularis externa) and individual protein expression responses (e.g. Lamc2 vs Bst2) are evident. In the muscularis externa, we found cytokine production as well as cell killing processes enhanced compared to the mucosa emphasizing its distinct role in the response against the S. Tm bacteria. Going beyond the control of bowel movement, the involvement of the muscularis externa is increasingly recognized by others (e.g. secretion of factors stimulating epithelial regeneration and proliferation⁷¹) and is in accordance with our finding showing a strong response to interferons. Interestingly, we found upregulation of individual proteases to be very distinct between the sub-tissue layers. A strong upregulation of proteases was observed with varying spatial distribution of MMPs in the inflamed intestinal tissue (e.g. MMP7 upregulated in epithelium, cathepsin D in the inflammatory infiltrate, others in both) in a published Salmonella fibrosis model⁷². This highlights that in acute (day 3) as well as in persistent Salmonella infection (day 21) the controlled spatial expression of proteases is important in the immune response and could potentially be used for development of targeted therapeutics.

One surprising finding in our study was the early onset, followed by a much more pronounced relaxation of fibronectin fibers in the muscularis externa compared to the delayed effects seen in the mucosa. The low tensional state fibronectin fibers in the smooth muscle layers are most probably linked to the shrinkage of the organ itself, which has been extensively reported previously^31,38,73. Cecal volume reduction must logically be associated with contraction of the smooth muscle layers. The performed pathway enrichment analysis of the N-terminomics dataset identified smooth muscle contraction as a significant term enriched in inflamed cecum (Fig. S5e), which further strengthens this link. Furthermore, proteases involved in extracellular matrix remodeling such as Cathepsin G and granzyme A, which exhibit capability to cleave fibronectin and other ECM fibers, were detected solely in the muscularis externa. Taken together, this discovery could hint towards the possibility of fibronectin fiber cleavage as a contributing factor to the strong FnBPA5 signal in the muscularis layers.

The observed substantial downregulation of metabolism-related proteins, upregulation of leukocyte-specific integrins as well as neutrophil markers specifically in the inflamed mucosa, but not in the muscularis externa, confirms the highly inflamed state of the mucosa. Our study revealed an unexpected distinct spatial pattern of fibronectin fiber relaxation in areas of neutrophil clusters within the mucosa, at the late stage of S. Tm infection (day 3). This finding raises additional questions about highly localized protease release and its functional consequences in the severely inflamed mucosal surrounding. The temporal dynamics observed, where neutrophil clusters are abundant on day 2, yet the FnBPA5 signal is only evident on day 3, suggest possible shifts in gene expression within these clusters leading to increased protease release targeting fibronectin. This might have interesting downstream functions as it is known that ECM fibers and more specifically also fibronectin fragments have important and different functions than their full-length counterparts^74–76. A different explanation could be that the tissue damage is increasing in these spatially defined areas due to granuloma-like structures and that the FnBPA5 signal is indicative for tissue destruction. Understanding the functional consequences of fibronectin fiber relaxation in neutrophil clusters is of particular interest, as stretched fibronectin fibers are indicative of healthy organs²⁸. Release of this tension may have implications locally or systemically, potentially impacting immune responses and pathogen clearance.

In summary, this study represents a significant step in unraveling the intricacies of cecal proteome and ECM dynamics during S. Tm infection on a sub-tissue layer basis. The synergistic approach of combining LCM with low biomass LC-MS/MS proteomics enriched by a mechanobiological perspective charts a novel trajectory for future investigations and providing a foundation for translational applications in infectious disease management.

Ethics statement. All animal experiments were approved by the legal authorities (license ZH120/19) Kantonales Veterinäramt Zürich, Switzerland) and performed according to the legal and ethical requirements. Animals were scored daily for any expected or unexpected adverse events. Humane endpoints were defined in the license. Special focus was put on the 3R principles (Replacement, Reduction and Refinement) for humane animal handling.

Mice. Male and female specific-pathogen-free C57BL/6J mice (10–11 weeks old) from an inbred colony at the ETH Phenomics center were used for all experiments. Animals were housed in groups of 2–5 animals in individually ventilated cages in the ETH Phenomics center (EPIC, RCHCI), ETH Zurich with ad libitum access of water and food at all times. Before the experiments mice were accustomed to their handling and trained to obtain a fruit-peanut mix (3:2:3 of 20% maltose solution, peanut-oil (sterilized), and fruit puree (fruit puree containing apples (56%), bananas (30% and raspberry (14%), pasteurized)) from the micropipette once per day on two consecutive days. One day before the S. Tm infection, the mice were pretreated with 25 mg of streptomycin in 75 µl sterile fruit-peanut solution via voluntary feeding. The Salmonella culture to infect the mice was prepared as overnight culture from wild-type Salmonella enterica serovar Typhimurium clone SB300, a derivative of strain SL1344 ³¹, grown for 12 h in Lysogeny Broth (LB) medium containing 50 µg/ml streptomycin at 37°C and 180 rpm. Then the bacterial culture was diluted 1:20 in fresh LB medium without antibiotics and the subculture was grown for 3 h at 37°C and 180 rpm. To prepare the fruit-peanut suspension, the bacteria were washed twice in cold PBS before adding. Mice were sacrificed by CO2 asphyxiation followed by blood withdrawal from the heart at the indicated time points and tissue samples were collected for further processing. The mock infected control group was sacrificed on day 3, together with the 72 h p.i. group. To avoid cage effects, the individual groups were formed by mice from multiple cages. During the infection period the animals were scored daily.

Analysis of S. Tm loads in intestines, mesenteric lymph nodes, spleens, and livers. Fresh fecal pellets, cecal content, spleen and liver samples were collected, weighed and placed in 1 ml of PBS. They then were homogenized by bead beating (3 mm steel ball, 25 Hz, for 1.5 min using a Tissue Lyser (Qiagen, Germany)). Numbers of CFU were determined by plating appropriate dilutions on Mc-Conkey agar plates (with streptomycin at 50 µg/ml) and incubating them overnight at 37°C.

Immunohistochemical procedure. Co-stainings for relaxed (Cy5.5-FnBPA5) and total fibronectin fibers as well as with anti-Ly6B.2 antibody (#MCA771G, BioRad, Hercules, California, USA) were performed as follows: Tissue cryosections (20 µm thick) on glass slides were encircled with a hydrophobic pen (H-4000, Vector Laboratories, USA) to decrease staining solution usage. For all following individual steps, 100 µl of solution was used per cryosection. The tissues were then washed once with PBS and blocked with 4% BSA in PBS for 30 min. Cy5.5-FnBPA5 solution was diluted in PBS, added to the sections (5 µg/ml) and incubated for 1 hour. The sections were then washed by immersing the whole glass slide into a beaker with PBS (3x 5 min each). In the next step, the tissue sections were fixed with 4% formaldehyde solution (#P087.3, ROTI®Histofix, Carl Roth) for 10 min and subsequently washed with PBS 3x for 5 min each. Then sections were blocked for 45 min using a blocking buffer containing 5% goat or donkey serum (depending on the host species of the secondary antibody) (#G9023 and #D9663, Merck) and 0.3 M glycine (#56-40-6, Merck) in PBS. Fibronectin was stained using a polyclonal anti-fibronectin antibody (ab23750, Abcam, Cambridge, UK) or anti-Ly6B.2 antibody at a dilution of 1:100 incubating over night at 4°C in a humidified chamber. After 3 washes with PBS (5 min each), the secondary antibody goat anti-rabbit IgG Alexa 488 (A11043, Thermo Fisher Scientific) or goat anti-rat IgG Alexa 488 (ab150157, abcam) at 1:200 was applied for 1 hour at room temperature. After a quick wash with PBS, co-stain with 4′,6-diamidino-2-phenylindole (DAPI) (D9564, Sigma Aldrich) was performed (10 µg/ml) for 10 min. Last, the sections were washed 3x for 5 min in PBS, dried and mounted using ProLong Gold antifade mounting medium (#P36930, Thermo Fisher Scientific). Mounted and stained sections were allowed to dry at room temperature and stored at 4° C before image acquisition.

Microscopy. All immunohistochemistry stains were imaged using the Nikon Eclipse Ti2 microscope, equipped with the Yokogawa Confocal Scanner Unit CSU-W1-T2 and operated with the NIS-Elements Software. Images were acquired using either a 20x 0.75 CFI Plan Apo λ or a 60x 1.2 CFI Plan Apo VC Water objective. SHG images were acquired using a Leica SP8 multi-photon microscope with an excitation at 880 nm.

Quantification of FnBPA5 signals. FnBPA5 signal was analyzed using a pixel-by-pixel approach. First, manual annotation in ImageJ and QuPath were made for muscosal and muscularis externa areas. Second, all pixels above the corresponding scrFnBPA5 signal were counted and their intensity was measured. For the analysis of FnBPA5-positive pixels, all xy coordinates plus intensity values were extracted from the images and separately treated for the mucosal and the muscularis externa areas. Then the percentage of positive pixels was calculated from all mucosal or muscularis externa pixels defined in the manual annotations. To test for robustness of the data, a bootstrapping algorithm was applied to test for the variability in the data. In detail, 10% of the data was selected 1000 times to calculate the internal error. Positive pixel counts as well as the intensity distribution were visualized using python. To calculate Ly6B.2-specific signal, a QuPath algorithm was used to detect Ly6B.2 clusters and then the FnBPA5-positive pixel coverage was calculated in these clusters versus the rest of the mucosa.

Mass spectrometry sample preparation and processing of LCM material. The MMI CellCut Laser Capture Microdissection device (Molecular Machines & Industries) was used to perform the Laser Capture Microdissection. 20 µm cryosections were captured on MMI membrane slides (MMI Prod. No. 50103) and subsequently stained with H&E using the MMI H&E Staining Kit Plus (MMI Prod. No. 70302). Collection of the microdissected tissue areas (in total 500.000 µm²) was done with the mmi isolation cap tubes (200 µl) and tissues were kept at -20° C until further processing. Tubes were turned upside down fixed in this position and the sample lysis was performed directly on the tube lid. Tubes were opened and 10 µl RIPA buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Cat# 89900 Thermo Scientific)) were added on top of the tissue on the lid itself. 1 µl of 10x TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, #C4706, Merck) was added to the lid and mixed carefully by pipetting up and down. 1 µl of CAA (400mM 2-Chloroacetamide, #C0267, Merck) was added, mixed carefully by pipetting and the tubes were closed to minimize evaporation. The tissues were incubated for 30 min at room temperature. Using a tabletop centrifuge the liquid was spun down and 48 µl ice-cold 100% acetone was added. This was followed by overnight incubation at -20°C. After acetone precipitation, the lids containing isolation caps were exchanged manually with normal lids to avoid detachment of the silicone inlets during high-speed centrifugation. Samples were then centrifuged for 5 min at 21.000 x g. The acetone was removed, and the samples were air dried in a fume hood. After that the samples were resuspended in 10 µl 4 M GdCl (Guanidinium Hydrochloride) in 50 mM HEPES (#G3272, Merck) and sonicated in a water bath sonicator for 10 min. This was followed by a dilution in 30 µl 50 mM HEPES (pH 8.5) for digestion, LysC was added in a 1:50 ratio and samples were incubated for 4 hours at 37°C. Afterwards samples were diluted in another 30 µl 50 mM HEPES (pH 8.5), trypsin was added in a 1:10 protease:protein ratio (w/w) and samples were incubated overnight at 37°C at 350 rpm. Using trifluoroacetic acid at a final concentration of 1% samples were acidified and the pH was verified using pH strips. After this the samples were centrifuged at 21.000 x g for 15 min and transferred onto EvoTip Pure trap columns for desalting and directly loading the samples onto the LC-MS. The EvoTip Pure tips were used according to the manufacturer’s instructions. In brief, Evotips were rinsed with 20 µl Solvent B (centrifugation at 800 g for 60 s). After this they were soaked in propanol until the Evotips turned pale white and equilibrated by soaking in 20 µl Solvent A (centrifugation at 800 g for 60 s). After loading the samples onto the wet Evotips, they were centrifuged again at 800 g for 60 s and washed with 20 µl Solvent A (centrifuged at 800 g for 60 s). Then, 100 µl Solvent A was added and the Evotips were centrifuged at 800 g for 10 s only.

Data independent acquisition mass spectrometry analysis. Samples were placed on the EvoSep One liquid chromatography system (EvoSep, Denmark) and measured in-line with an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) coupled to a FAIMSpro device. Peptides were loaded on a EV1106 C18 column (15cm x 150 µm, 1.9 µm diameter) and separated with the Whisper100 nanoflow and a 20SPD (samples per day) method consisting of a 58-minute gradient. Eluting peptides were injected to the mass spectrometer using a 20 µm fused silica emitter (EV1087), at a static voltage of 2300 V, carrier gas flow of 3.6 L/min and 240°C ion transfer tube temperature and a positive polarity. A single compensation voltage of -45 V was applied to the FAIMS device during acquisition with a high-resolution MS1 (HRMS1) data-independent acquisition method. MS1 scans were recorded in the orbitrap detector at a 120.000 resolution, with a scan range of 400–1000 m/z, normalized AGC target of 300%, and injection time set to automatic. MS2 scans were recorded over the full m/z range with an isolation window of 8 m/z and 1 m/z window overlap. Peptides were fragmented using HCD (high collision dissociation), with a fixed normalized collision energy of 32%. The orbitrap resolution was set to 60.000 with first mass of 200 m/z. Normalized AGC target was set to 1000%, with maximum injection time set to automatic. MS1 scans were interspersed every 24 scan events (loop count 24), splitting the m/z range in three equal, 200 m/z parts. The raw data were searched with DIA-NN version 1.8, using a library-free (directDIA) approach and MS1 level quantification. The reference proteome was the mouse proteome database obtained from Uniprot (UP000000589 reviewed, accessed 17/06/2022). Precursor FDR was set to 1%, while Met N-terminal excision, Met oxidation and C carbamidomethylation were added as modifications. Match between runs and RT-dependent cross-run normalization were set to True. Trypsin/P was used as the protease, with one allowed missed cleavage, with otherwise default settings. Search results and protein quantification tables were used for further post-processing and analysis. To note, protein groups are denoted as proteins throughout the text, aiming to enhance the fluidity of reading.

TMT-TAILS sample preparation. The cecum samples used were embedded in cryomedium for other experimental design reasons and first had to be cleaned thoroughly from the freezing medium. First, 500 µl of homogenization buffer (4 M GuHCl, 0.1M EDTA, 1:10 protease inhibitor in MilliQ) was added to the samples and samples were placed in a Bioruptor® Pico sonication device (Diagenode, Hologic Inc, Beglium) for 45 cycles (30 sec on, 30 sec off). Then ice-cold TCA (trichloroacetic acid) was added to a final concentration of 20% and incubated for 20 min on ice. Samples were centrifuged at 16.000 g for 20 min at 4°C and the supernatant was discarded. Then, 4 washes with ice-cold 10% TCA were performed (centrifugation for 5 min at 16.000 g to pellet the samples in-between). Subsequently, the samples were washed with 1 ml ice-cold 100% acetone for another 4 times in total. After the last wash, the samples were air-dried. After this they were resuspended in 20 µl of 0.2 M NaOH and incubated for 2 min at room temperature with 350 rpm. Then, 80 µl TAILS buffer (250 mM HEPES, 2.5 M GuHCl) was added to the samples and the pH was measured to match a pH of 7.8. Further sample processing was performed as published in a detailed methods paper⁷⁷. In short, reduction of cysteine residues was performed by adding TCEP to a final concentration of 5 mM and incubation of samples at 65°C for 45 min with 400 rpm. Then, cysteine residues were alkylated by addition of CAA to a final concentration of 20 mM and another incubation step at 65°C at 400 rpm for 30 min. To prepare TMT reagent, 200 µg aliquots were dissolved in 110 µl of DMSO and mixed with equal amount of sample by pipetting. Samples were incubated for 1 h at room temperature. Then, to quench the labeling reaction ammonium bicarbonate was added to a final concentration of 100 mM and mixed by vortexing, followed by 30 min incubation at room temperature. Subsequently, all labeled samples with the individual TMT reagents were mixed in a 15 ml conical tube. Then, 8 times the sample volume of ice-cold acetone and 1 time the sample volume of ice-cold methanol were added and the samples were incubated at -80°C for at least 2 hours. Then, the samples were centrifuged at 4500 x g at 4°C for 20 min and the supernatant was discarded. For further washing 5 ml ice-cold methanol was added and again centrifuged at 4500 g at 4°C for 20 min. After discarding the supernatant, the samples were air-dried. The samples were then resuspended in 100 mM NaOH at a ratio of 10:1 (w/v) protein to NaOH and mixed well by pipetting. Then, MilliQ water was added at protein:MilliQ ratio of 5:4 (w/v) and 1 M HEPES at a ratio of 10:1 (w/v). The samples were subsequently transferred into low binding tubes. For digestion, trypsin was added at protein:trypsin ratio of 50:1 (w/w) and the samples were incubated overnight (at least 16 h, max. 24 h) at 37°C with 350 rpm. 10% of the digest was taken out as non-pullout sample and stored at -20°C until desalting. For the remaining samples the pH was adjusted to pH 6.5 using 2 M HCl and the HPG-ALD polymer was added in a protein:polymer ratio of 1:4 (w/w). Then, cyanoborohydride was added to a final concentration of 50 mM and samples were incubated at 37°C for 16 h. On the next day 30 kDa Amicon Ultra-0.5 ml centrifugal filter units were prepared with 400 µl MilliQ water following the manufacturer’s instructions and centrifuged at room temperature. Then, the samples were transferred to the filter unit and centrifuged at 10.000 x g for 10 min at RT. The filters were washed with 100 µl of 100 mM ammonium bicarbonate and centrifuged again at 10.000 g for 10 min at RT. Both flowthroughs were combined, acidified with TFA to a final concentration of 1% TFA and further processed together with the NPO sample which was brought to a final volume of 200 µl using resuspension buffer (2% ACN, 1% TFA). To prepare the desalting cartridges, the Sep-Pak got activated with 800 µl of methanol. Then, washed with 800 µl of elution buffer (80% ACN, 0.1% formic acid (FA), 19.9% MilliQ grade water) and equilibrated with 800 µl of equilibration buffer (3% ACN, 1% TFA) twice. Then the samples were loaded, and the cartridges were washed twice with 800 µl of washing buffer (0.1% formic acid). After addition of 200 µl Buffer B (ACN) the flowthroughs were collected, and the eluates were dried in a speed vacuum concentrator. Subsequently, the dried samples were resuspended in resuspension buffer and measured for peptide concentration. 500 ng of peptides were loaded on EvoTips similarly to LCM samples for subsequent LC-MS/MS analysis.

TMT-TAILS mass spectrometry analysis. We measured the pre-TAILS and TAILS samples, containing the total proteome and the enriched N-terminome respectively, with the EvoSep One platform (Evosep, Denmark) in line with an Orbitrap Exploris 480 mass spectrometer, which was coupled to a FAIMSpro interface. Peptides were separated using a PepSep column (Bruker Daltonics, cat. no. 1895812) during an active gradient of 118 minutes (Whisper100 10SPD method). Peptides were injected into the mass spectrometer with a PepSep emitter (Bruker Daltonics, cat. no. 1893519) with a positive ion spray voltage of 2300 V, and an ion transfer tube temperature of 240°C. The carrier gas flow was set to 3.6 L/min, and FAIMS was operated in standard resolution in 3 different compensation voltages (CVs) of -40, -55, and − 70. Data acquisition was performed as follows across all CVs. MS scans were acquired in the Orbitrap at 120,000 resolution and a scan range of 375–1500, automatic maximum injection time, RF Lens of 60%, and normalized AGC target of 300%. We included filters of charge state between 2–7, dynamic exclusion with a duration of 60 seconds with 10 ppm mass tolerance, minimum intensity of 5000 and precursor fit at 70% before MS/MS acquisition. Top 10 data dependent MS/MS scans were acquired for each CV as centroids in the Orbitrap, which was operated at 60,000 resolution. We used an isolation window of 0.7 m/z, first mass of 110, HCD fragmentation at 34% NCE, with automatic maximum injection time and normalized AGC target at 75%.

The raw data were analyzed with Proteome Discoverer v2.4. Both raw files were included in the same search as fractions and searched against the mouse proteome, using the same database as the LCM analysis. The peptide library was constructed with semi-tryptic specificity and 2 maximum missed cleavages. Methionine oxidation (+ 15.995) and asparagine deamidation (+ 0.984) were added as dynamic modifications. TMTpro (+ 304.207) and acetylation (+ 42.011) were added as dynamic modifications on the peptide and protein terminus, respectively. Cysteine carbamidomethylation (+ 57.021) and lysine TMTpro labeling were added as static modifications. Peptide-spectrum matching was performed with Sequest HT, and FDR control with Percolator with 1% strict, and 5% relaxed FDR cutoffs. Peptides and proteins were quantified using the Reporter Ion Quantifier node, normalized to the total peptide amount of each channel. The peptide group and protein reports were exported for downstream analysis with Python 3.8 and CLIPPER 2.0.

Data and material availability. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁷⁸ partner repository with the dataset identifier PXD043256. The reviewer account has the following credentials: username - [email protected] , password: agLJiVDZ. Materials available on request to the corresponding author.

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Competing interest reported. V.V. is a co-founder and scientific advisory board member of Tandem Therapeutics AG (CHE-192.070.977), an early-stage start-up focused on the development of extracellular matrix targeting drugs. All other authors declare no conflict of interest.

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Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

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Abstract

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Introduction

Results

Spatial proteomics analysis of healthy and severely inflamed mouse ceca

Functional analysis of proteins detected solely in the inflamed conditions

The distribution of identified matrisome proteins is consistent across both sub-tissue layers and disease states

Probing healthy cecal tissue using a fibronectin fiber tension probe

Discussion

Materials and Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1