Pyroptosis mediated by gasdermins (GSDMs) plays crucial roles in infection and inflammation. Pyroptosis triggers the release of inflammatory molecules, including damage-associated molecular patterns (DAMPs). However, the consequences of pyroptosis—especially beyond interleukin (IL)-1 cytokines and DAMPs—that govern inflammation are poorly defined. Here, we show intercellular propagation of pyroptosis from dying cells to bystander cells in vitro and in vivo. We identified extracellular vesicles (EVs) released by pyroptotic cells as the propagator of lytic death to naive cells, promoting inflammation. DNA-PAINT super-resolution and immunoelectron microscopy revealed GSDMD pore structures on EVs released by pyroptotic cells. Importantly, pyroptotic EVs transplant GSDMD pores on the plasma membrane of bystander cells and kill them. Overall, we demonstrate that cell-to-cell vesicular transplantation of GSDMD pores disseminates pyroptosis, revealing a domino-like effect governing disease-associated bystander cell death.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and have become significant players in immune system functioning, primarily by facilitating cell-to-cell communication. Immune cells like neutrophils and dendritic cells release EVs containing bioactive molecules that modulate chemotaxis, activate immune cells, and induce inflammation. EVs also contribute to antigen presentation, lymphocyte activation, and immune tolerance. Moreover, EVs play pivotal roles in antimicrobial host defense. They deliver microbial antigens to antigen-presenting cells (APCs), triggering immune responses, or act as decoys to neutralize virulence factors and toxins. This review discusses host and microbial EVs' multifaceted roles in innate and adaptive immunity, highlighting their involvement in immune cell development, antigen presentation, and antimicrobial responses.

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1β maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance. Kumari et al. show that host-derived extracellular vesicles capture systemic LPS and transfer it to the cytosol of immune cells via CD14-dependent endocytosis, triggering caspase-11-mediated gasdermin D activation and pyroptosis.

Type I interferons (IFNs) are consequential cytokines in antibacterial defense. Whether and how bacterial pathogens inhibit innate immune receptor-driven type I IFN expression remains mostly unknown. By screening a library of enterohemorrhagic Escherichia coli (EHEC) mutants, we uncovered EhaF, an uncharacterized protein, as an inhibitor of innate immune responses including IFNs. Further analyses identified EhaF as a secreted autotransporter—a type of bacterial secretion system with no known innate immune-modulatory function—that translocates into host cell cytosol and inhibit IFN response to EHEC. Mechanistically, EhaF interacts with and inhibits the MiT/TFE family transcription factor TFE3 resulting in impaired TANK phosphorylation and consequently, reduced IRF3 activation and type I IFN expression. Notably, EhaF-mediated innate immune suppression promotes EHEC colonization and pathogenesis in vivo. Overall, this study has uncovered a previously unknown autotransporter-based bacterial strategy that targets a specific transcription factor to subvert innate host defense.

Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis1,2. Among GSDMs, GSDMB is unique owing to its distinct lipid-binding profile and a lack of consensus on its pyroptotic potential3-7. Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity4. Shigella, an intracellular, human-adapted enteropathogen, evades this GSDMB-mediated host defence by secreting IpaH7.8, a virulence effector that triggers ubiquitination-dependent proteasomal degradation of GSDMB4. Here, we report the cryogenic electron microscopy structures of human GSDMB in complex with Shigella IpaH7.8 and the GSDMB pore. The structure of the GSDMB-IpaH7.8 complex identifies a motif of three negatively charged residues in GSDMB as the structural determinant recognized by IpaH7.8. Human, but not mouse, GSDMD contains this conserved motif, explaining the species specificity of IpaH7.8. The GSDMB pore structure shows the alternative splicing-regulated interdomain linker in GSDMB as a regulator of GSDMB pore formation. GSDMB isoforms with a canonical interdomain linker exhibit normal pyroptotic activity whereas other isoforms exhibit attenuated or no pyroptotic activity. Overall, this work sheds light on the molecular mechanisms of Shigella IpaH7.8 recognition and targeting of GSDMs and shows a structural determinant in GSDMB critical for its pyroptotic activity.

The noncanonical inflammasome, comprising inflammatory caspases 4, 5, or 11, monitors the cytosol for bacterial lipopolysaccharide (LPS). Intracellular LPS-elicited autoproteolysis of these inflammatory caspases leads to the cleavage of the pore-forming protein gasdermin D (GSDMD). GSDMD pore formation induces a lytic form of cell death known as pyroptosis and the release of inflammatory cytokines and DAMPs, thereby promoting inflammation. The noncanonical inflammasome-dependent innate sensing of cytosolic LPS plays important roles in bacterial infections and sepsis pathogenesis. Exciting studies in the recent past have significantly furthered our understanding of the biochemical and structural basis of the caspase-4/11 activation of GSDMD, caspase-4/11's substrate specificity, and the biological consequences of noncanonical inflammasome activation of GSDMD. This review will discuss these recent advances and highlight the remaining gaps in our understanding of the noncanonical inflammasome and pyroptosis.

Interferons are potent antimicrobial effectors and thus an attractive target for pathogen interference. In this issue of Cell, Alphonse et al. reveal that the Shigella effectors OspC1 and OspC3 employ a surprising mechanism to block interferon signaling and attenuate antibacterial responses, thus securing their replicative niche.

Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1β, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance.

We investigated the impact of nutrient intake on hydration biomarkers in cyclists before and after a 161 km ride, including one hour after a 650 mL water bolus consumed post-ride. To control for multicollinearity, we chose a clustering-based, machine learning statistical approach. Five hydration biomarkers (urine color, urine specific gravity, plasma osmolality, plasma copeptin, and body mass change) were configured as raw- and percent change. Linear regressions were used to test for associations between hydration markers and eight predictor terms derived from 19 nutrients merged into a reduced-dimensionality dataset through serial k-means clustering. Most predictor groups showed significant association with at least one hydration biomarker: 1) Glycemic Load + Carbohydrates + Sodium, 2) Protein + Fat + Zinc, 3) Magnesium + Calcium, 4) Pinitol, 5) Caffeine, 6) Fiber + Betaine, and 7) Water; potassium + three polyols, and mannitol + sorbitol showed no significant associations with any hydration biomarker. All five hydration biomarkers were associated with at least one nutrient predictor in at least one configuration. We conclude that in a real-life scenario, some nutrients may serve as mediators of body water, and urine-specific hydration biomarkers may be more responsive to nutrient intake than measures derived from plasma or body mass.

Endurance athletes commonly strive for optimal hydration status during and after events, and have vast nutrition options available to support performance and well-being. PURPOSE: We aimed to evaluate relationships among nutrients consumed during exercise and markers of hydration status. METHODS: Fifty-one cyclists (age mean=51y and range=21-72y; 49 males, 2 females) completing a 161km event (mean=26°C, 76%RH; maximum=30°C, 93%RH) recorded all dietary intake during the ride. Five hydration markers (urine color and specific gravity, plasma osmolality (Posm), plasma copeptin (Pcop), and body mass change (BM) were collected before and after (POST) the ride, and one hour after a 650mL water bolus (POST1h). Linear regressions tested associations between hydration markers and eight predictor terms derived from nineteen nutrients merged into a reduced-dimensionality dataset through serial k-means clustering. As an indicator of water retention signaling, Pcop tertiles were analyzed via two-way ANOVA to evaluate nutrient intake influence. RESULTS: Five predictor clusters were significantly associated to hydration markers (number of associated hydration markers in parenthesis): 1) glycemic load + carbohydrates + sodium (one), 2) protein + fat + zinc (one), 3) magnesium + calcium (two), 4) pinitol (three), and 5) water (four); caffeine, potassium, fiber, betaine, and three sugar-alcohols did not associate with hydration markers. All hydration markers (except Posm) associated to at least one nutrient predictor. Pcop POST tertiles (13.5±5.9, 34.4±7.4, and 76.8±40.0 pmol/L, respectively) differed by sodium (1st vs. 3rd tertile p=0.0047; 2217±1295 and 1747±1214 mg, respectively) and water intake (1st vs. 3rd and 2nd vs. 3rd, all p<0.0001; 1st=4910±1722, 2nd=4887±1011, 3rd=3837±1097 g). Pcop POST1h tertiles (7.4±3.0, 22.0±5.4, and 54.2±36.7 pmol/L, respectively) differed by water intake (1st vs. 3rd and 2nd vs. 3rd, all p<0.0001; 1st=4921±1652, 2nd=4953±1063, and 3rd=3759±1072 g). CONCLUSION: These data suggest that some nutrients impact fluid-electrolyte balance and hydration markers. Nutrient intake appears to mediate urinary markers more than Pcop, and Pcop more than BM. Further, sodium and water appear to best mitigate water retention signaling following exercise and rehydration.

Changes in plasma osmolality (Posm) and arginine vasopressin (AVP)-mediated signaling regulate thirst and drinking behavior. Copeptin is a peptide derivative of the AVP preprohormone and thought to be more stable and measureable than AVP as a biomarker of the hydration process. PURPOSE: This investigation aimed to evaluate hydration biomarkers, including copeptin, responses to exercise-induced dehydration and partial rehydration. METHODS: Fifty-two registrants (mean age: 52y, range: 21-72) in a 161km cycling event under warm and humid environmental conditions (mean = 26°C, 76%RH; maximum = 30°C, 93%RH) participated. Posm, urine specific gravity (Usg), urine color (Ucol), thirst, and plasma copeptin were measured at 3 time points: before (PRE) and shortly after (POST) the ride, and one hour following a 650mL water bolus at ambient temperature consumed in 6 increments within 3min (POST1h). Subjects consumed their typical diet during, but were not permitted to eat or drink between ride completion and the 1h post period. RESULTS: Subjects lost 2.2 ± 1.1% body mass at POST, and all variables significantly increased from PRE to POST (Posm = 295.8 ± 3.9 to 299.1 ± 5.6 mOsm·kg-1; U = 1.017 ± 0.005 to 1.021 ± 0.006; Ucol = 3 ± 1 to 5 ± 2; copeptin = 7.50 ± 4.9 to 42.23 ± 35.74 pmol·L-1; thirst = 3 ± 1 to 5 ± 2; all p < 0.05). At POST (body mass = -2.0 ± 1.1%) Posm returned to PRE (294.4 ± 5.7 mOsm·kg-1), while U (1.021 ± 0.006), Ucol (5 ± 2), and thirst (5 ± 2) remained elevated compared to PRE (all p < 0.05). sg Copeptin remained elevated at POST1h vs. PRE (p < 0.0001) but decreased from POST to POST1h (POST1h = 27.87 ± 28.57 pmol·L-1; p < 0.0001). CONCLUSION: Well-studied hydration biomarkers and the more recent biomarker copeptin tracked exercise-induced dehydration (PRE vs. POST), but differed in response to partial rehydration with a 650mL bolus (POST1h): copeptin tracked with partial rehydration, while Posm would indicate adequate and urine markers inadequate fluid replacement occurred. Ongoing work includes analyses of the relationship between food intake during the ride and hydration biomarkers to determine factors that may contribute to change magnitude at POST and differences in biomarker responses at POST1h. Grant Funding: University of Hartford faculty grant; University of Connecticut ONSF, faculty start-up funds, and OUR

The heat shock protein (HSP) response is critical to the cellular stress response. Circulating, extracellular stress‐inducible HSP72 is known to respond to exercise‐heat stress, but little is known about other, constitutively expressed and fundamental members of the HSP family. The aim of this study was to investigate the extracellular HSP27, HSP60, and HSP90α response during a prolonged cycling event in hot and humid conditions. Seventy‐six subjects (mean±SD: age, 51.64±10.47 years; height, 176.44±6.85 cm; mass, 86.78±13.93 kg) were recruited at the Hotter'n Hell 100 mile cycling event in Wichita Falls, Texas. Blood and urine samples, as well as data on body weight and gastrointestinal temperature (TGI) were recorded before (pre) and after (post) the event. Plasma was isolated from blood samples, stored at −80°C, and later analyzed with StressMarq Biosciences ELISAs for HSP27, HSP60, and HSP90α concentrations. Student's t‐test was used to test for significant differences of means. TGI significantly increased from pre to post (pre: 37.14±0.55 °C; post: 38.12±0.79 °C; p<0.05). HSP27 and HSP90α were both significantly elevated post race (pre27: 2.73±0.51 ng × mL−1; post27: 3.46±0.73 ng × mL−1; pre90α: 18.03±3.27 ng × mL−1; post90α: 21.53±4.84 ng × mL−1; p < 0.05). However, there was no significant difference in HSP60 plasma concentration from pre to post (pre: 44.05±7.38; post: 43.65±6.81; p=0.77). In conclusion, significant increases in circulating HSP27 and HSP90α suggest that chaperones required for thermotolerance and protein degradation or folding during heat stress increase in extracellular compartments post exercise‐heat stress. However, our data suggest that chaperones specifically involved in mitochondrial protein assembly or molecular danger signals like HSP60 are not elevated in blood fractions. Ongoing work will characterize cellular expression of these proteins in circulating peripheral blood mononuclear cells.Support or Funding InformationNew investigator start‐up, McNair Scholar ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise in stressful environmental conditions induces multiple independent signaling pathways that result in inflammatory cytokine release. Immune responses that cause and are driven by the systemic release of inflammatory and pyrogenic cytokines contribute to endotoxemia, sepsis, and other immunity‐related exertional heatstroke (EHS) pathophysiology. Our preliminary data from the Falmouth Road Race (FRR, 11.3 km, Falmouth, MA), a race, known to have high incidence (15–20 annually) of EHS, suggest that at least one particular pro‐inflammatory pathway stimulated by circulating lipopolysaccharide (LPS) is robustly upregulated post‐race. In this study, we aimed to test the hypothesis that competing in a high intensity running road race in a hot and humid environment will result in increased cytokine levels that may serve as biochemically downstream predictive measures of an EHS susceptibility marker, core temperature. We recruited 32 (2015), 33 (2016), and 32 (2017) subjects (43±12 years, 174.2±8.6 cm, 72.3±12.6 kg, 17.0±5.1% body fat, 43.49±8.01 mL·kg−1·min−1 VO2max) participating in the FRR. We measured environmental and physiological variables, and acquired venous blood pre (PRE) and immediately post (POST) race. We quantified six different cytokines (IL‐6, IL‐8, IL‐10, IL‐1β, TNF‐α, and IL‐12p70) in anti‐coagulant treated blood plasma (pg·ml−1). ANOVA with Bonferroni post hoc tests and Pearson correlations were calculated to determine statistically significant (p<0.05) differences in cytokine concentration and correlations between cytokine concentrations and POST gastrointestinal temperature (Tgi). POST (vs. PRE) Tgi was elevated significantly each year and for the pooled 3‐year data (mean±SD °C, PRE: 36.97±0.48, POST: 39.55±0.79, p<0.00001). Pooled (3‐year) IL‐6 (mean±SD, PRE: 29.25±17.45), IL‐10 (PRE: 18.54±11.95), and IL‐8 (PRE: 53.99±22.56) were increased at POST (IL‐6: 51.41±44.56, IL‐10: 29.55±22.02, IL‐8: 64.99±28.21). POST (vs. PRE) cytokine responses were consistent within each year (p<0.05) and with pooled, 3‐year data (p<0.0001). Although analysis of individual years suggested that PRE markers such as TNF‐α (p<0.019) and IL‐12p70 (p<0.0066) positively correlated with elevated post race gastrointestinal temperatures, analysis of the pooled 3‐year data revealed that PRE cytokine values are not significantly correlated with POST Tgi. Overall, we observed that resting cytokine levels did not identify any chronic inflammation present in subjects who experienced markedly high POST Tgi or exertional heat illness. Ongoing work will elucidate whether post race cytokine values, relative change in individual cytokine responses, or other mechanisms of inflammatory signaling correlate to exertional heat illness incidence and/or abnormally elevated core temperature.Support or Funding InformationFalmouth Road Race, McNair Scholars Program (UConn), CICATS, KSIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Previous investigations at the Falmouth Road Race (FRR; Falmouth, MA) indicated that combined exercise, heat, and dehydration stress increased gastrointestinal tract permeability. This subsequently exposed the circulatory system to components of the microbiome usually found only in the gut. Lipopolysaccharide (LPS), a component of Gram‐negative bacterial membranes, is one such element released into circulation, especially post‐exercise. LPS in circulation activates host immune cell signaling cascades that can lead to inflammatory responses (e.g. cell pyroptosis, exertional heat stroke, and sepsis) and upregulated proinflammtory gene expression (e.g. TNF‐α and IL‐1β). While subjects of the 2015 FRR (N=30) had elevated core temperature post race compared to pre (pre 36.87°C vs. post 39.87°C, p<0.05), there is unexplained variation in the relationship among the level of circulating LPS (2.90EU/ml pre vs. 3.48EU/ml post, p<0.05; Hycult Biotech, ELISA), core temperature, and subsequent heat stroke presentation. Since some strains of bacteria induce a strong immune response while others do not, it is of interest to determine if structural variations in LPS contribute to varying immune system activation during exercise‐heat stress. Diet influences the composition of the microbiome and different microbiome species have varying LPS structures. Because of this, even short‐term alterations in macronutrient consumption can alter the composition and gene expression of the microbiome. Ongoing research will utilize extraction of LPS from pre‐race and post‐race plasma and HPLC/MS to characterize structural variations in LPS in individuals of the 2015, 2016, and 2017 FRR.Support or Funding InformationNew investigator start‐up, McNair Scholar Program, FRRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influenza strain diversity and variability and individual immune health contribute to reduced vaccine efficacy. Among adjuvants that enhance vaccine effectiveness, several studies have examined the relationship between acute exercise stress and influenza vaccination immune responses in humans. It is proposed that acute bouts of stress enhance immune response, but optimal intervention, timing of intervention, and mechanisms have yet to be identified. As Toll‐like receptor (TLR) agonists enhance innate immune responses and support development of chronic humoral responses, exercise induced release of the TLR4 ligand lipopolysaccharide (LPS) into circulation may be a mechanism for benefit of pre‐vaccine exercise. This study aimed to assess the effects of exercise and exercise‐heat stress completed before seasonal influenza vaccination (IV) on short‐ and long‐term immune responses. Thirty‐four males (body mass, 77.41±12.48kg; age, 21.5±4.4y; maximal aerobic capacity (VO 2max ), 43.53±8.24mL/kg/min) were randomized into 1 of 3 groups: no exercise (NE, n=12) or 60 minutes of 60%VO 2max exercise with (MEH, n=11) or without (ME, n=11) environmental heat stress. Exercise and baseline blood samples were completed 25.11±5.92h before IV, and subsequent blood samples were collected 23.45±6.13h, 13.06±2.96d, and 29.54±3.73d following IV. Exercise environmental conditions were different between ME and MEH, respectively (average wet bulb globe temperature, 32.91±2.67°C, 41.21±2.88°C; p<0.001), but physiological stress was similar (average rectal temperature, 37.30±0.48°C, 27.61±1.36°C, p=0.513; heart rate, 114±105bpm, 118±105bpm, p=0.103) due to controlled exercise intensity. On‐going research will utilize hemagglutinin inhibition, cortisol, and LPS assays to quantify virus‐specific antibody responses, stress, and TLR4 activation respectively. Understanding innate and humoral responses will elucidate the potential of exercise and exercise‐heat stressors as adjuvants for IV. Support or Funding Information New investigator start‐up, InCHIP Interdisciplinary Seed Grant This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Lipopolysaccharide (LPS) is a gram‐negative bacterial marker that might be released into circulation during exercise‐heat stress. LPS induces an innate immune response by binding LPS‐binding protein (LBP) and its cognate receptor, TLR4 (toll‐like receptor 4), expressed on CD16+CD14+ circulating immune cells. LPS‐induced TLR4 activation results in robust immune responses which drive endotoxemia, sepsis, and putatively, exertional heatstroke pathophysiology. PURPOSE We aimed to test the hypothesis that competing in prolonged cycling (50–100 mile event) or high‐intensity running (7 mile race) events in hot and humid environments (heat index>80°F) result in LPS‐induced TLR4 activation and changes in cellular gene expression. METHODS We recruited 64 subjects participating in the Hotter n' Hell Hundred (HHH) ultraendurance cycling event (Wichita Falls, TX) and 36 subjects racing in the Falmouth (FAL) Road Race (Falmouth, MA). We sampled venous blood pre (PRE) and immediately post (IP) each event, and also 30 minutes after IP (30IP) in FAL. We isolated circulating PBMCs (peripheral blood mononuclear cells) which were stained for surface markers CD14, CD16, and TLR4 using fluor‐conjugated antibodies. We used multi‐color digital flow cytometry on‐site to analyze cell populations. We also isolated anticoagulant‐treated plasma samples from whole blood for analysis of circulating LPS (Hycult Biotech, ELISA) and LBP (R&D ELISA) concentrations. At both events, we measured environmental and physiological variables. We statistically analyzed data using repeated measures ANOVA (LSD post hoc, α‐level:p≤0.05) for differences (PRE vs. IP, PRE vs. 30IP). RESULTS Circulating LPS increased in both HHH riders and FAL racers by 25% (p<0.05) and 39% (p<0.04), respectively. By 30IP, FAL racers' circulating LPS levels had returned to PRE values (p>0.19). In both races, circulating LBP remained unchanged (p>0.33). CD14+CD16+ cells expressing TLR4 in circulation were significantly increased at IP (p<0.05). CONCLUSION We conclude that circulating LPS is increased post‐exercise heat stress and analysis of downstream activation is required to determine role of LPS in exercise‐heat stress. Support or Funding Information We would like to thank Connecticut Institute for Clinical and Translational Science and McNair Scholars Program for funding the research and thank Hotter'N Hell Hundred and Falmouth Road Race for their support.