key: cord-0292716-2glkstrm
authors: Malka, Orit; Malishev, Ravit; Bersudsky, Marina; Rajendran, Manikandan; Krishnamohan, Mathumathi; Shaik, Jakeer; Tikhonov, Evgeni; Sultan, Eliya; Koren, Omry; Apte, Ron N.; Rosental, Benyamin; Voronov, Elena; Jelinek, Raz
title: Tryptophol acetate and tyrosol acetate, metabolites secreted by a probiotic yeast, halt cytokine storm
date: 2021-12-17
journal: bioRxiv
DOI: 10.1101/2021.12.16.472991
sha: 99327283329189799e5e5d4d88fc71d3bead286c
doc_id: 292716
cord_uid: 2glkstrm

Probiotic fermented foods are perceived as contributing to human health and capable of protecting against inflammation, however solid mechanistic evidence for the presumptive therapeutic benefits is lacking. Here we report that tryptophol acetate and tyrosol acetate, small molecule metabolites secreted by the probiotic milk-fermented yeast Kluyveromyces marxianus exhibit remarkable anti-inflammatory properties. Comprehensive in vivo, ex vivo and in vitro experiments, employing LPS-induced ‘cytokine storm’ models, reveal dramatic effects of the two molecules, added in tandem, on mice morbidity, laboratory parameters and mortality. In parallel, significant attenuation of pro-inflammatory cytokines including IL-6, IL-1α, IL-1β and TNF-α, and reduction of reactive oxygen species were recorded. Importantly, tryptophol acetate and tyrosol acetate did not completely suppress cytokine generation, but rather brought their concentrations back to baseline levels, further maintaining core immune functions, including phagocytosis. The anti-inflammatory effects of tryptophol acetate and tyrosol acetate were mediated through downregulation of TLR4, IL-1R, and TNFR signaling pathways and increased A20 expression, attenuating NF-κB level. In addition, the two molecules had a significant impact on mice microbiome, increasing the abundance of the genus Bactericides, known to exhibit anti-inflammatory properties. Overall, this work illuminates pronounced and broad-based immune modulation properties of probiotic yeast-secreted metabolites, uncovering their mechanism of action and underscoring potential new therapeutic avenues for severe inflammation.

Sepsis is a life-threatening condition manifested by severe inflammation leading to 21 multiple organ dysfunction. The inflammatory host response associated with both 22 innate and adaptive immunity mechanisms play an important role in the development 23 of the clinical and pathological manifestations of sepsis 1 . Recently, it has been found 24 that the prognosis of the disease is dependent not only on the virulence of the 25 microorganisms, but, mainly, on the host response affected by the pathogen-associated 26 molecular patterns (PAMPs) 2,3 . Recruitment of immune cells and secretion of soluble 27 mediators by the cells can exacerbate the severity of the disease 4 . Specifically, the 28 release of pro-inflammatory molecules, such as interleukin-6 (IL-6), tumor necrosis 29 factor-alpha (TNF-α) and interleukin-1α and 1β (IL-1α and IL-1β), trigger "cytokine 30 storms", systemic inflammation often leading to multi-organ failure and adverse 31 clinical outcomes with high mortality rates 5 . Cytokine storms occur in various disease 32 conditions, including sepsis and septic shock 6 and acute stages in chronic diseases 7 . 33 Severe inflammation and the occurrence of cytokine storms were also shown to be 34 major causes of mortality from COVID-19 8, 9 . 35 Despite the significant progress in inflammation treatment following the discovery of 36 antibiotics, high mortality from sepsis still exists. Thus, new approaches to improve 37 conventional therapies are highly sought. Varied food products have been touted to 38 endow anti-inflammatory properties and as such attract significant interest 10 . Food- 39 extracted substances have been particularly explored as anti-inflammatory agents, 40 including probiotics 11, 12 , curcumin 13 , resveratrol 14 , plant extracts 15 , and phenolic 41 compounds from natural sources 16 . However, the therapeutic benefits of most such 42 substances against severe inflammation have been limited 17 , and, moreover, detailed 43 mechanistic understanding of their perceived anti-inflammatory properties are 44 generally lacking. 45 Probiotics, particularly milk-fermented microorganism mixtures (yogurt, kefir), have 46 been known to bolster the innate immune system and host-defense mechanisms against 47 pathogens 18, 19 . In a recent study, we reported on a yet-unrecognized mechanism for 48 cross-kingdom inhibition of pathogenic bacterial communication and virulence by a 49 small molecule -tryptophol acetate -secreted by the probiotic yeast Kluyveromyces 50 marxianus in a milk-fermented probiotic microorganism mixture 20 . Specifically, 51 tryptophol acetate was found to disrupt biofilm formation and reduce virulence of 52 several human pathogenic bacteria, underscoring a novel mechanism for combating 53 bacterial colonization and pathogenicity. 54 Here, we report that tryptophol acetate and tyrosol acetate, another K. marxianus -55 secreted metabolite, exhibit remarkable anti-inflammation activities, in in vitro, ex vivo 56 and in vivo models. Through application of LPS-induced cytokine storm, we observed 57 that the two molecules had synergistic anti-oxidation, anti-inflammatory, clinical, 58 histological and hematological systemic protective effects against severe inflammation. 59 Importantly, tryptophol acetate and tyrosol acetate did not give rise to immune system 60 shutdown, rather reduced pro-inflammatory cytokine production to baseline levels 61 while retaining core immune processes including phagocytosis and generation of anti- 62 inflammatory cytokines. Detailed molecular analysis indicates that the anti-63 inflammatory activities of tryptophol acetate and tyrosol acetate are mediated through 64 downregulation of TLR4, IL-1R, and TNFR signaling pathways and suppression of NF- 65 κB activity. In particular, we discovered that the molecules enhanced expression of 66 A20, a key modulator of NF-κB signaling pathways 21 . Overall, this study demonstrates 67 remarkable anti-inflammatory properties of probiotic yeast-secreted metabolites and 68 furnishes a detailed mechanistic description of the effects, underscoring their 69 significant therapeutic potential. contributing to toxic effects in diverse diseases and pathological conditions 23 . Figure 1 85 depicts application of a spectrophotometric assay monitoring the visible absorbance of 86 the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which is widely employed for 87 evaluating the anti-oxidation activity of biomolecules 24 . In this assay, the DPPH free 88 radical, stable at room temperature, is reduced in the presence of an antioxidant 89 molecule, giving rise to a colorless ethanol solution. The bar diagram in Figure 1A 90 shows the degree of quenching of DPPH radical absorbance (and concomitant anti-91 oxidative properties) by tryptophol acetate and tyrosol acetate, when added individually 92 as well as together. Figure 1A indicates a dramatic synergistic anti-oxidation effect for 93 the mixture of the two molecules. Specifically, while 1 individually gave rise to 10% 94 quenching of DPPH absorbance and 2 alone reduced the absorbance by ~3%, when the 95 1 and 2 were added together (at the same concentrations) to the DPPH solution, 30% 96 absorbance attenuation was recorded ( Figure 1A ). The results of the oxygen radical absorbance capacity (ORAC) assay in Figure 1B However, upon co-addition of 1 or 2, the fluorescence decays were significantly longer, 117 up to around 400 min (in the case of 1, purple curve) and 800 min (for 2, beige curve), 118 likely accounting for scavenging of the peroxyl radical by the molecules. Strikingly, 119 significant inhibition of peroxyl-induced fluorescence quenching occurred when 120 tryptophol acetate and tyrosol acetate were added together to the solution (green curve 121 in Figure 1B) , underscoring their synergistic anti-oxidation effect.

Oral uptake of tryptophol acetate and tyrosol acetate mixture provides protection 124 against LPS-induced cytokine storm in mice 125 As tryptophol acetate and tyrosol acetate exhibit significant synergistic anti-oxidation 126 activity, we next investigated the anti-inflammatory properties of the 1+2 mixture in 127 vivo using a model of lipopolysaccharide (LPS)-induced inflammation, which triggers 128 a massive pro-inflammatory cytokine release identified as a "cytokine storm" 26, 27 . In 129 the experiments, C57BL/6 mice were injected with LPS (30 mg/kg), and the effects of 130 the 1 + 2 mixture administrated orally by gavage (each molecule at 150 μg/Kg per 131 mouse) were monitored up to 156 hours after LPS injection (scheme in Figure 2A ). the severe inflammation (i.e. the "cytokine storm"), 100% of the mice orally given the 146 mixture of 1 + 2 survived ( Figure 2B ). Furthermore, the LPS-administered mice 147 experienced substantial weight loss prior to mortality ( Figure 2C , black line). In 

To prove the therapeutic effects of the molecules, we carried out another experiments, 152 in which 1+2 mixture was given to mice that were already impacted by the onset of interval, severe signs of the disease were already evident, including decreased motor 156 activities, ruffled fur, diarrhea, substantial eye discharge and respiratory distress.

Notably, the results in Figure 2D demonstrate that while all untreated mice died within 158 48 hours after LPS injection, 100% of the mice treated with the 1 + 2 mixture survived.

The weight lost patterns in mice treated with the molecules 28 hours after LPS 160 administration when severe inflammation was already developed ( Figure 2E ) were 161 similar to the case of mice that were orally given the two molecules simultaneously 162 with LPS (e.g., Figure 2C ). Overall, the results presented in Figure 2 the healing effects of tryptophol acetate and tyrosol acetate (e.g., Figure 2 ) also lead to leading to enhanced pulmonary vascular leakage 32, 33 . Figure 229 Figure 3B indicates that 1+2 mixture also prevented tissue damage in the liver. The

H&E staining image of healthy liver is presented in Figure 3B , top row. In contrast, the 231 structure of liver tissue was significantly damaged 6 and 24 hours after LPS 

inflammation. 249 We further assessed the effects of the tryptophol acetate and tyrosol acetate mixture 

310 reducing phagocytosis function 311 To investigate the mechanistic basis of the clinical and biological immunomodulation 312 effects of tryptophol acetate and tyrosol acetate (e.g., Figures 1-4) , we performed ex- 313 vivo experiments using murine peritoneal macrophages. Figure receptors.

Accounting for the reduction of the upstream receptors in the NF-κB pathways by the 415 tryptophol acetate and tyrosol acetate (e.g., Figure 6B ), we further tested the effects of 416 the molecules upon expression of the A20 protein, known to suppress signaling 417 cascades associated with the TLR receptors 40 (Figure 6C-D) . The bar diagram in Figure   418 6C presents experimental data corresponding to RAW 264.7 cells treated with 1+2 419 mixture and the A20 m-RNA levels measured after 30 min and 60 min. As shown in 420 Figure 6C , both LPS, and the 1+2 mixture co-added with LPS, induced significant 421 expression of A20 gene within 60 minutes after addition to the cells. However, when 422 1+2 were co-added to the cells together with LPS, significantly higher expression of 423 A20 was found.

To assess the protein level of A20, we further carried out WB analysis ( Figure 6D ), Figure 7E underscores the effect of treating the LPS-administered mice with tryptophol 478 acetate and tyrosol acetate. Importantly, the abundance of Bacteroides increased in the 479 mice that were treated with 1+2 compared to the mice that were not treated (24 hours 480 after LPS injection; Figure 7E ). This taxon was also abundant in the microbiome of 481 healthy mice (ANCOM test significance W=68 and W=72 respectively (Table S1) ).

This result is important as Bacteroides has been linked to varied immune-protective 483 and anti-pathogenic activities associated with microbiome modifications 44 . Figure 6A ).

Furthermore, the RT-PCR data in Figure 6 attest to pronounced downregulation of the 573 main upstream receptors involved in NF-κB activation -TLR4, IL-1R and TNFR.

These observations are mechanistically significant as it is known that production and 575 secretion of pro-inflammatory cytokines are governed by the TLR/NF-κB signaling 576 pathway, perceived as a major "gateway" cascade in innate immunity, and are 577 associated with the pathogenesis of severe inflammation that promotes lung, liver, and 578 intestinal injuries 44 . For example, binding of LPS to TLR-4 was shown to induce lung 579 parenchymal damage, neutrophil accumulation in the interstitial and alveolar 580 compartments, elevated vascular permeability and pulmonary edema 53, 54 .

Further molecular insight upon the underlying immunomodulatory mechanism 582 of tryptophol acetate and tyrosol acetate was furnished by analysis of A20 protein levels 583 ( Figure 6 ). A20 is a prominent negative feedback regulator of NF-κB signaling. It has 584 been reported that mice genetically deficient in A20 develop severe inflammation, 585 underscoring the central role of A20 in suppression of NF-kB-dependent inflammation 586 and tissue homeostasis 55 . Importantly, we found that the tryptophol acetate and tyrosol 587 acetate mixtures significantly induced, in both gene and protein levels, the expression 588 A20 (Figure 6 C,D) . Thus, our data suggest that the protection furnished against severe 589 inflammation by tryptophol acetate and tyrosol acetate may be mediated by triggering 590 A20 expression and concomitant inhibition of the TLR4/IL-1R/TNFR-NF-κB pathway.

Due to our observations that administration of tryptophol acetate and tyrosol 592 acetate orally gave rise to the pronounced anti-inflammatory effects, we assessed the 593 effects of the molecules on the gut microbiota (Figure 7) . The relationship between 594 severe inflammation and microbial dysbiosis has been extensively studied in recent 595 years 56, 57 . The gut microbiota have been shown to enhance host immunity to pathogens, 596 and dysbiosis has been linked to increased susceptibility of severe inflammation 58 . 597

Accordingly, we characterized the gut microbial compositions of mice prior and after 

Oxygen radical absorbance capacity (ORAC assay) 676 The ORAC method is based on the oxidative degradation of the fluorescent molecules 677 after mixing with a free radical generator, for example, azo compounds. This method 678 determines the ability of the sample to neutralize short-lived free radicals. The assay 

Immunoreactive protein bands were visualized using the enhanced chemiluminescent.

Data analysis 884 All results are expressed as the mean ± SD or mean ±SE, as indicated. Data for all in-885 vivo and in-vitro experiments were analyzed by a one/two-way ANOVA followed by 886 Tukey post-hoc test using GraphPad Prism (GraphPad Software, San Diego, Ca). A p-887 value of ≤ 0.05 was considered statistically significant .

Fecal microbiome analysis 890 Total DNA was extracted from fecal samples using a PureLink microbiome DNA 891 extraction kit (Invitrogen, Carlsbad, CA) followed by a 2-min bead-beating step, as 892 previously described by Shouval, R. et al. 64 

Inflammatory responses and inflammation-associated diseases in 933 organs

Targeting cytokines, pathogen-associated molecular 935 patterns, and damage-associated molecular patterns in sepsis via blood purification

Recognition of pathogen-associated molecular patterns by 938 TLR family

T helper type cytokines in sepsis: time-shared variance and 940

correlation with organ dysfunction and hospital mortality

Biomarkers of cytokine storm as red flags for severe and fatal 943

COVID-19 cases: A living systematic review and meta-analysis

Sepsis and septic shock Richard

Chronic inflammation in the etiology of disease across the life span

Targeting inflammation and cytokine storm in COVID-19

The signal pathways and treatment of cytokine storm in

All foods affect health': Understandings of functional foods and healthy 954 eating among health-oriented Finns

Effect of probiotics on biomarkers of cardiovascular disease: 956 Implications for heart-healthy diets

Gut brain axis: Diet microbiota interactions and 958 implications for modulation of anxiety and depression

A review of its effects on human health

Health benefits and 963 molecular mechanisms of resveratrol: A narrative review

The Benefits of Plant Extracts for Human Health

Food Phenolic Compounds: Main Classes, Sources and Their 966

Oxidative Stress and Chronic Degenerative Diseases -A Role for 967

Oxidative Stress Chronic Degener

Risks and safety of 970 polyphenol consumption

Administration of kefir-fermented milk protects mice against 972

Giardia intestinalis infection

Immunomodulating capacity of kefir

Cross-kingdom inhibition of bacterial virulence and communication 976 by probiotic yeast metabolites

Modulating 978 inflammation through the negative regulation of NF-κB signaling

Microorganism mixtures

Reactive oxygen species: The dual role in physiological and 984 pathological conditions of the human body

Development and validation of an 987 improved oxygen radical absorbance capacity assay using fluorescein as the 988 fluorescent probe

Physiological aspects of Toll-like receptor 990

activation in sepsis-induced acute kidney injury

Experimental models of Gram-negative sepsis. 22-30

Cytokine release syndrome

LPS-induced CD11b+Gr1intF4/80+ 996 regulatory myeloid cells suppress allergen-induced airway inflammation

Insulin enhances leukocyte-endothelial cell adhesion in the retinal 999 microcirculation through surface expression of intercellular adhesion

Neutrophils and acute lung injury

Mobilization and Margination of Bone Marrow Gr-1 high 1003

Monocytes during Subclinical Endotoxemia Predisposes the Lungs toward Acute 1004

Lung-marginated monocytes modulate pulmonary microvascular 1006 injury during early endotoxemia

12/15-Lipoxygenase Orchestrates the Clearance of Apoptotic Cells 1009 and Maintains Immunologic Tolerance

Inflammatory pathways 1011 in female reproductive health and disease

Reactive oxygen 1015 species in inflammation and tissue injury

The nuclear factor-k B ( NF-k B ) family of 1018 transcription factors

TLR-4, IL-1R and TNF-R signaling to NF-κB: Variations on a 1020 common theme

The role of pattern-recognition receptors in innate immunity

Update on toll-like receptors

Efficient selection for high-expression 1024 transfectants with a novel eukaryotic vector

A20 inhibits both the degradation and limited 1026 processing of the NF-κB p105 precursor: A novel additional layer to its regulator role

Linking the Human Gut Microbiome to Inflammatory Cytokine 1029

Isolation of anti-inflammatory and epithelium reinforcing 1031 bacteroides and parabacteroides spp. From a healthy fecal donor

The pathogenesis of sepsis and potential therapeutic 1033 targets

Phenotypic characterization of alveolar monocyte recruitment in 1035 acute respiratory distress syndrome

Toll-like receptors in sepsis-associated cytokine storm and their 1038 endogenous negative regulators as future immunomodulatory targets

Sepsis-associated severe interleukin-6 storm in critical coronavirus 1041 disease 2019

ROS-dependent signal transduction

Reactive 1045 oxygen species in metabolic and inflammatory signaling

The effect of cyclosporine and 1048 dexamethasone on an alveolar macrophage cell line (NR8383)

Nimbolide protects against endotoxin-induced acute respiratory 1051 distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear 1052 translocation

Distinct 1054 roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung 1055 injury

CD14 controls the LPS-induced endocytosis of toll-like receptor 4

Failure to regulate TNF-induced NF-kappaB and cell death responses 1059 in A20-deficient mice

Gut microbiota and inflammation

The Relationship Between Gut Microbiota and 1063

Inflammatory Diseases: The Role of Macrophages

Role of the Microbiota in Immunity and inflammation Yasmine

Intestinal interplay of quorum sensing molecules and human 1067 receptors

Ameliorates Colon Inflammation in Preclinical Models of Crohn's Disease

Bacteroides thetaiotaomicron and Faecalibacterium 1072 prausnitziiinfluence the production of mucus glycans and the development of goblet 1073 cells in the colonic epithelium of a gnotobiotic model rodent

Unravelling the collateral damage of antibiotics on gut bacteria

Bioactive components, antioxidant, and anti-inflammatory activities of 1079 the wood of Albizia myriophylla

Patterns of salivary microbiota injury and oral mucositis in 1081 recipients of allogeneic hematopoietic stem cell transplantation

Reproducible, interactive, scalable and extensible microbiome data 1084 science using QIIME 2

Greengenes, a chimera-checked 16S rRNA gene database and 1086 workbench compatible with ARB

DADA2: High-resolution sample inference from Illumina 1088 amplicon data