Mucosal-associated invariant T (MAIT) cells are reduced and dysfunctional in acute melioidosis

Mucosal-associated invariant T (MAIT) cells are reduced and dysfunctional in acute melioidosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mucosal-associated invariant T (MAIT) cells are reduced and dysfunctional in acute melioidosis Fazle Rabbi Chowdhury, Martha Zewdie, Priyanka Abraham, Srija Moulik, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9083519/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Burkholderia pseudomallei (BP), the causative agent of melioidosis, is a major cause of sepsis in Southeast Asia, especially in people with diabetes mellitus (DM). The role of Mucosal-associated invariant T (MAIT) cells; innate-like T cells important for antibacterial immunity; in melioidosis is unknown. We measured MAIT cell activation by BP in vitro using co-culture assays with THP-1 cells, and evaluated MAIT cell frequency, activation, and function ex vivo in an observational cohort (n = 120) of melioidosis patients and endemic controls with and without DM in Thailand. We show that BP induces IFN-γ secretion by MAIT cells in a cytokine dependent manner. In acute melioidosis, circulating MAIT cells, particularly the double-negative (DN) subset, were significantly reduced, and highly activated but dysfunctional, with reduced IFN-γ responses to BP and E. coli which were restored upon recovery. Among acute patients, non-survivors showed lower granzyme B and IFN-γ expression. Acute melioidosis patients with DM co-morbidity exhibited reduced DN MAIT cell frequency and responses to E. coli compared to non-DM patients. Overall, the frequency and function of MAIT cells is impaired during acute melioidosis, especially in patients with DM, indicating a key role for these cells in antibacterial defence and disease susceptibility. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology mucosal associated invariant T cells sepsis melioidosis diabetes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection 1 resulting in millions of deaths each year 2 , 3 . Bacterial infections are responsible for a major proportion of sepsis cases in the world, and among them Gram-negative bacteria are the main culprit 4 , 5 . Some of the commonest causative Gram-negative organisms are, Escherichia coli , Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enterica serotype Typhi 6 . In addition, the Gram-negative bacterium Burkholderia pseudomallei (BP) which causes the neglected tropical disease melioidosis is an important cause of adult sepsis in Asia 4 , 5 . With an estimated incidence of 165,000 annual cases worldwide 7 and an in-hospital case fatality rate of up to 50% in some regions of Thailand 8 , melioidosis causes a significant burden in endemic regions. Several risk factors including diabetes mellitus (DM), chronic kidney and lung disease, malignancy and excessive alcohol use are associated with melioidosis 9 . DM constitutes the biggest risk factor with a 12-fold increased risk of melioidosis, and over 50% of melioidosis patients have this metabolic disease 10 . We and others have previously demonstrated a role for T cells 11 and natural killer (NK) cells 12 , 13 in survival from melioidosis in humans, as well as differences in immunological pathways associated with survival in patients with DM co-morbidity 14 . Recent studies in mouse models point towards an important contribution of unconventional T cells to protection. In the absence of invariant NKT (iNKT) cells, survival time was reduced in B. pseudomallei infected BALB/c mice, and early activation of this cell subset contributed to bacterial clearance and enhanced survival 15 . In a C57B6/J mouse model of pulmonary melioidosis the presence of γδT cells was important for survival via a reduction of neutrophil-induced inflammation in the lungs 16 . Mucosal-associated invariant T (MAIT) cells are another unconventional T cell subset with both innate and adaptive properties 17 . This cell population plays an important role in human immune responses to bacterial and viral infections 18 . MAIT cells are abundant in the gut, liver, lung and blood, comprising 1–10% of peripheral blood T lymphocytes 19 . In humans, MAIT cells express a conserved invariant T cell receptor (TCR) α-chain (Vα7.2-Jα33) and are defined by their restriction to the non-classical MHC class I-related (MR-1) molecule 20 , 21 . Their natural ligands are derived from folic acid (vitamin B9) and from an intermediate in the microbial biosynthesis of riboflavin (vitamin B2) 22 . Therefore, organisms which synthesise riboflavin precursors (Gram-negative bacteria, Yeasts) can stimulate MAIT cells in an MR-1-dependent manner 21 . MAIT cells can also be activated by cytokines in a TCR-independent manner. Notably, IL-12 and IL-18 are potent inducers of MAIT effector functions 23 . Established mouse models demonstrate a beneficial role of MAIT cells in immune defence against several bacterial pathogens including K. pneumoniae 24 , Francisella tularensis 25 , Legionella longbeachae 26 and extraintestinal pathogenic E. coli 27 . In humans with severe sepsis and septic shock, an early reduction of MAIT cell numbers can be observed compared to healthy controls 27 , 28 . The role of MAIT cells in melioidosis has not been explored before. In this study we characterise the ability of BP to activate MAIT cells and how MAIT cell frequency and function is affected in a cohort of acute melioidosis patients from Thailand. We further assess the contribution of DM co-morbidity on the properties of these important effector cells. RESULTS B. pseudomallei activates MAIT cells in vitro in a cytokine dependent manner We first assessed the ability of BP to induce IFN-γ secretion from MAIT cells in a previously established co-culture assay 29 , 30 using healthy PBMC and THP-1 cells incubated with PFA-fixed BP at 25, 50 and 75 bacteria per cell (BpC) (Fig. 1 A). MAIT cells were phenotypically characterised by the expression of Vα7.2 and high expression of CD161 in T cell subsets (Fig. 1 B). For this analysis we focused on MAIT cells within the CD8 + T cell subset as this constitutes the majority of MAIT cells in human blood 31 , 32 . BP induced IFN-γ secretion from circulating CD8 + MAIT cells, with the strongest induction cytokine expression at BpC 50 (50 BP per THP1 cell). Incubation with 50 BP per THP1 cell was used in all subsequent experiments (Fig. 1 C). In comparison to E. coli , used at the previously optimised dose of BpC 30 25 , BP induced three-fold lower levels of IFN-γ expression in CD8 + MAIT cells (Fig. 1 D). Notably, secretion of IL-12 by THP-1 cells in response to fixed E.coli was 3-fold higher compared to that induced by fixed BP ( E.coli : 2500 pg/ml, BP: 782 pg/ml). Blocking of both IL-12 and IL-18 during the incubation of BP with THP-1 cells completely abrogated IFN-γ expression (median: 1.4, IQR: 0.5–2.4% IFN-γ positive) compared to the isotype control (median: 10.7, IQR: 5.3–11.1% IFN-γ positive, p = 0.0149), indicating that IFN-γ production was predominantly cytokine-dependent. Blocking of MR-1 only marginally reduced cytokine production, and there was no synergistic effect of blocking both cytokines and MR-1 in this culture system (Fig. 1 E). This demonstrates that BP is able to activate MAIT cells in a predominantly cytokine-dependent manner at the assessed timepoint. Circulating MAIT cells are reduced and dysfunctional in acute melioidosis Having demonstrated that MAIT cells are activated by BP in vitro we next sought to study the frequency, activation and function of MAIT cells ex vivo in the context of acute melioidosis infection. We used samples from a cohort of acute and recovered melioidosis patients recruited in Thailand between 2012–2017. Individuals with and without DM from the same region were used as endemic controls (Fig. 2 A). Acute melioidosis patients were slightly but significantly older (median 59, IQR 51–65 years, p = 0.016) than recovered patients (median 53, IQR 45–61 years) and healthy controls (median 52, IQR 48–61 years). There were no differences in sex, the proportion of individuals with DM, or history of renal impairment between groups. We found a 2.7-fold reduction in frequency of circulating MAIT cells within the double negative (DN) T cell compartment in acute melioidosis cases (median 2.8, IQR 0.9–4.7%) compared with healthy controls (median 7.5, IQR 2.0-11.8%, p = 0.0059) (Fig. 2 B ). Importantly, one year after infection the frequency of DN MAIT cells in recovered patients (median 6.7, IQR 4.2–11.3%) returned to levels comparable to healthy controls (Fig. 2 B). The same effect was observed when comparing the frequency of MAIT cells in the overall live cell compartment ( Supplementary Fig. 1 ). In contrast, the frequency of CD8 + MAIT cells remained unaffected during acute disease. Next, we assessed activation and functional properties of MAIT cells. Due to the low numbers of MAIT cells in acute disease we combined CD8 + and DN MAIT cell compartments in order to increase events for detection of downstream parameters by flow cytometry. MAIT cells (CD8 + and DN combined) were highly activated (CD69+) in acute melioidosis (median 49.1, IQR 35.5–60.1%) compared with healthy controls (median 11.6, IQR 3.7–23.2%, p < 0.0001) and recovered patients (median 4.7, IQR 2.5–15.4%, p < 0.0001). MAIT cells displayed higher GzmB expression in acute disease (median 8.6, IQR 5.5–27.5%) compared with healthy controls, (median 3.3, IQR 1.7–15.8%) although this difference was not statistically significant (p = 0.0979) (Fig. 2 C). Furthermore, IFN-γ production by MAIT cells from acute melioidosis patients following in vitro stimulation was abrogated compared with healthy controls. Responses to BP were significantly reduced (median, IQR: Acute 0.4, 0-1.2%; Healthy 6.1, 1.6–13.5%, p = 0.0006) and albeit not reaching statistical significance we also noted lower reactivity to E. coli (median, IQR: Acute 1.0, 0.2–8.4%; Healthy 11.0, 3.6–19.4%, p = 0.0580). Upon recovery, IFN- γ expression in response to BP returned to levels comparable with the control group (Fig. 2 D, Supplementary Fig. 2 ). To assess whether a reduced capacity for IFN-γ expression in MAIT cells is a particular feature of melioidosis or a more common effect in sepsis we measured MAIT cell IFN-γ expression in a small cohort of sepsis patients with different disease aetiology in Bangladesh. MAIT cell IFN-γ expression upon stimulation with the respective causative pathogen was significantly impaired in patients infected with K. pneumoniae and E. coli ( Supplementary Fig. 3B, C ) and a trend for reduced levels was observed in those infected with S. Typhi ( Supplementary Fig. 3A ) compared with healthy controls. Overall, our data highlights depletion of MAIT cells in the DN T cell compartment in acute melioidosis, and shows that despite being highly activated in acute disease, MAIT cells are limited in their ability to produce IFN-γ in response to bacterial stimulation in vitro . Table 1 Participant characteristics for melioidosis cases (acute and recovered) and endemic controls. Melioidosis Acute Recovered Control p-value* N = 55 N = 24 N = 41 Age 0.016 Median age 59 53 52 Age range 33–84 25–88 33–73 Interquartile range 51–65 45–61 48–61 Sex, n (%) 0.45 Female 16 (29%) 8 (33%) 17 (41%) Male 39 (71%) 16 (67%) 24 (59%) 28-Day Mortality, n (%) NA Survived 30 (55%) 24 (100%) 41 (100%) Died 25 (45%) 0 (0%) 0 (0%) Co-Morbidities, n (%) Diabetes 31 (56%) 12 (50%) 22 (54%) 0.87 History of renal impairment 11(23%) 5 (21%) 0 (NA%) > 0.99 *Pearson Chi-Squared test; Kruskal-Wallis rank sum test; Fisher's exact test Impaired GzmB and IFN-γ expression in MAIT cells of non-survivors We have previously shown that IFN-γ secretion by conventional T cells is impaired in acute melioidosis patients who died compared to those who survived 11 . To address whether a similar effect is observed for MAIT cells we stratified the acute melioidosis cohort based on clinical outcome ( Supplementary Table 2 ). While frequency and activation (CD69+) of MAIT cells were comparable in patients who survived and those who died within 28-days of hospital admission (Fig. 3 A and B ), GzmB expression (Fig. 3 B) and IFN-γ production (Fig. 3 C) in response to BP were reduced in non-survivors (GzmB median, IQR: Survived 19.5, 6.3–29.2%; Died 7.3, 2.6–12.2%, p = 0.0564; IFN-γ median, IQR: Survived 0.7, 0.3–1.5%; Died 0.0, 0.0-1.2%, p = 0.0404). This shows a further impairment of MAIT cell function in patients with fatal outcome. MAIT cell frequency and function are reduced in acute melioidosis patients with DM Since DM is an independent risk factor for melioidosis, we compared the frequency and activation of MAIT cells in acute and recovered melioidosis patients between those with and without DM ( Supplementary Table 3 ). Acute melioidosis patients with DM showed a 50% reduction in the proportion of MAIT cells within the DN T cell compartment (median 2.1, IQR 0.6–3.2%) compared with the non-DM group (median 4.4, IQR 2.0-7.3%, p = 0.0136). No difference was observed for the CD8 + MAIT cell subset (Fig. 4 A). MAIT cell expression of CD69 and GzmB was not significantly different in acute patients with DM compared to those without but we note that GzmB expression is slightly elevated in the DM group (Fig. 4 B). Importantly, IFN-γ production by MAIT cells in response to E. coli was significantly impaired in acute melioidosis patients with DM (median 0.3, IQR 0.0-2.4%) compared to those without DM (median 5.1, IQR 1.0-11.2%, p = 0.0181, Fig. 4 C). In contrast. recovered patients did not show differences in cell frequency, activation or function based on DM status (Fig. 4 D-F) indicating that MAIT cell dysfunction in DM is context dependent. Association of MAIT cell features with clinical parameters In order to assess the combined impact of clinical and demographic factors on MAIT cell frequency and activation we performed GLM analysis using age, sex, DM status, 28-day mortality and history of renal impairment as covariates. In line with univariate analysis, we identified DM as being significantly associated with a reduced proportion of MAIT cells within the DN T cell compartment and 28-day mortality being associated with lower expression of GzmB in MAIT cells (Table 2 ). In contrast, DM was independently associated with significantly higher expression of GzmB in MAIT cells when taking relevant covariates into account. Differences in CD69 expression on MAIT cells were not associated with any of the tested variables. Table 2 Generalized linear model testing the association of MAIT frequency and activation with demographic and clinical parameters Variables Estimates Dependent Predictor Coef SE Coef p value DN MAIT % of T cells Age 0.023 0.015 0.121 Sex (male) 0.141 0.333 0.672 28-day Mortality (yes) -0.514 0.311 0.098 Diabetes (yes) -0.566 0.285 0.047 History of renal impairment (yes) 0.574 0.332 0.084 CD69% of MAIT cells Age 0.006 0.007 0.374 Sex (male) 0.116 0.126 0.359 28-day Mortality (yes) 0.116 0.132 0.381 Diabetes (yes) -0.135 0.133 0.313 History of renal impairment (yes) 0.146 0.132 0.265 GzmB % of MAIT cells Age -0.001 0.024 0.971 Sex (male) -0.185 0.383 0.629 28-day Mortality (yes) -0.854 0.410 0.037 Diabetes (yes) 1.070 0.426 0.012 History of renal impairment (yes) 0.340 0.416 0.413 Gamma log linked, Coef=Coefficient, SE=standard error DISCUSSION This is the first study to characterise the MAIT cell response in the context of the neglected tropical disease melioidosis. Here we show that BP efficiently induces IFN-γ secretion by Vα7.2 + CD161hi MAIT cells in an overnight (20 hours) co-culture system with THP-1 cells. In our hands, blocking of cytokine signalling using antibodies targeted at the receptors for IL-12 and IL-18 completely abrogated the expression of IFN-γ in MAIT cells with no further reduction achieved by additional blocking of MR-1. Ussher et al 30 previously demonstrated a time dependent increase in IFN-γ production by MAIT cells stimulated with E. coli , with early IFN-γ secretion (5 hours) being entirely dependent on MR-1, and late IFN-γ secretion (20 hours) dependent on MR-1 in combination with IL-12 and IL-18. Similarly, C. difficile -induced IFN-γ production was found to be initially MR-1 dependent, with a more pronounced role for cytokines in driving IFN-γ production and cytotoxic features after longer incubation times 33 . In contrast, our data does not provide evidence for combined action of MR-1 and cytokines in induction of MAIT cell IFN-γ in response to BP after overnight culture, suggesting predominantly TCR-independent activation. The extent to which MAIT cells secrete IFN-γ is dependent on the levels of MR-1 upregulation, TLR stimulation and induction of IL-12 in antigen presenting cells (APC) 34 . E. faecalis , which is a weak inducer of IL-12 in THP-1 cells, also induced weaker MAIT IFN-γ responses compared to E coli , suggesting that MAIT cell activation and cytokine release are pathogen-specific 23 . In line with this, we show that BP compared with E. coli is a weaker inducer of IL-12 secretion by primed THP-1 cells, likely resulting in lower induction of IFN-γ by MAIT cells. In a cohort of melioidosis patients in Thailand, we show that MAIT cell frequency is significantly reduced during acute infection, with the cells that are present being highly activated but dysfunctional. We observed dampened IFN-γ production by MAIT cells in response to BP and E.coli , with responses returning to normal upon recovery. Within the acute melioidosis group we did not find evidence for differences in MAIT cell frequency and activation based on outcome of infection, but impaired function including reduced GzmB expression and IFN-γ production was noted in patients who died within 28-days of hospital admission. Our findings align with other studies of MAIT cells in acute bacterial disease. Marked specific depletion of MAIT cells was found in acute non-streptococcal septic patients compared to healthy controls, and this was associated with susceptibility to nosocomial infection 28 . Similar findings have been found in inflammatory and infectious diseases including acute cholecystitis 35 , pulmonary tuberculosis (TB) 36 , 37 and scrub typhus 38 . In a recent S . Typhi human challenge study, CD8 + MAIT cell frequency was sharply decreased early post-infection in volunteers who developed typhoid disease compared to those resistant to disease 39 . Reduced circulating MAIT numbers in infection could be due to recruitment of the cells into sites of infection, or increased apoptosis in the case of sepsis. As demonstrated and reviewed by Hotchkiss et al 40 , extensive apoptosis of immune cells including T cells, B cells and dendritic cells is a hallmark of sepsis. Zheng et al have recently shown that MAIT cells are reduced in circulation but enriched in tissue during acute appendicitis 41 . Interestingly, the reduction in MAIT cells in acute melioidosis was specific to the DN T cell compartment and not the CD8 + T cell subset. Notably, DN MAIT cells have been shown to exhibit higher apoptotic gene signatures and they are more prone to activation-induced apoptosis 42 possibly explaining the cell subset specific depletion observed in our study. In acute bacterial infection, there is evidence of diminished functional capacity of circulating MAIT cells 43 . MAIT cell IFN-γ production has been previously shown to be reduced during active mycobacterial infection 36 , which is in line with our findings. We identified specific changes in MAIT cells during acute melioidosis in patients with DM, including reduced proportion of DN MAIT cells, increased GzmB expression and reduced IFN-γ production upon stimulation with bacteria. DM, the major risk factor for melioidosis, is associated with chronic inflammation, reduced T cell function, and dysfunction of the innate immune system; specifically neutrophil and monocyte function 44 . MAIT cells have been previously shown to be reduced in frequency and function in blood and adipose tissue of Type 2 DM (T2DM) patients and obese individuals 45 , 46 . Magalhaes et al have shown that MAIT cells from T2DM patients have a pro-inflammatory phenotype characterised by higher levels of IL-17, IL-2, IFN-γ and GzmB compared to healthy individuals when activated with PMA/Ionomycin in vitro 45 . In contrast, MAIT cells were less activated upon specific TCR activation and showed a skewed cytokine response away from IFN-γ and TNF towards an IL-17 biased response in T2DM compared to healthy controls 45 . A subset of IL-17 secreting MAIT cells has also been associated with various chronic inflammatory conditions 47 . Our study shows similar impairment of MAIT cell function in the context of acute bacterial infection with reduced IFN-γ secretion in response to E. coli in patients DM co-morbidity. Although beyond the scope of this study, it will be interesting to follow up the IL-17 bias in the context of melioidosis and DM. Overall, our results point toward specific functional alterations of MAIT cells in people with DM during acute melioidosis infection. MAIT cell function was generally low in acute melioidosis with a further reduction seen in those who died from the disease. Future studies are needed to specifically address the contribution of MAIT cells to disease susceptibility in people with DM and their role in early control of bacterial infections such as melioidosis. Limitations The number of MAIT cells was highly reduced in acute disease therefore analysis of downstream properties of MAIT cells was restricted to samples with enough cells. We characterised MAIT cells by their expression of CD161 and Vα7.2 and did not use 5-OP-RU loaded MR1 tetramers for a more direct identification of MAIT cells. We focussed on the measurement of IFN-γ as the main effector molecule produced by MAIT cells upon activation. Measuring IL-17 especially in the context of DM co-morbidity and local immune responses in the lungs might give further insights into the role of MAIT cells in disease susceptibility and melioidosis pathogenicity. Methods Patient recruitment We used a patient cohort recruited into an observational study of acute melioidosis at Sunpasitthiprasong Hospital, Ubon Ratchathani, Thailand between 2012-2017 11,12 . Patients over 18 years with culture-confirmed melioidosis at a median of 5 days after admission to hospital (IQR 4–6 days) 11 were included in the study. 67% of patients had a diagnosis of DM (defined for the purpose of this study as a past medical history of diabetes and/or a blood glycated haemoglobin (HbA1c) of ≥ 7%) 12,48 . 28-day mortality was determined using hospital mortality records and follow-up telephone calls. Recovered melioidosis patients were seen one year after initial enrolment (week 52). Uninfected individuals with and without DM were recruited as endemic controls. Gram-negative culture-confirmed sepsis cases were recruited as part of the Aetiology of Fever study at Dhaka Medical College Hospital (DMCH) and Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM) Hospitals, Dhaka, Bangladesh. Adult patients of 18 years or more who were admitted with a history of fever ≥ 38 degrees for more than 48 hours were included. Acute Gram-negative sepsis patients (defined according to SIRS criteria) 49 were admitted 1–2 weeks into the course of their illness. Healthy endemic control subjects were recruited from the blood transfusion department of DMCH, and critically ill non-infectious patients (acute stroke, acute pesticide poisoning, diabetic ketoacidosis, severe acute asthma and acute exacerbation of COPD) were enrolled from the medicine department at DMCH. Healthy controls from the UK were recruited as part of the GI Biobank study at the University of Oxford and samples were used for MAIT in vitro assays including blocking experiments. Ethical approvals The ethics committees of the Faculty of Tropical Medicine, Mahidol University (TMEC-12-014 and TMEC-15-046), of Sunpasitthiprasong Hospital, Ubon Ratchathani (018/2555 and 017/2559) and the Oxford Tropical Medicine Research Ethics Committee (OXTREC; 64 − 11 and 35 − 15) approved the Thai melioidosis study protocol. The ethics committee of DMC (MEU-DMC/ECC/2015/97), BIRDEM Hospital (BADAS-ERC/EC/17/0127), and OXTREC (OXTREC: 51 − 16) approved aetiology of febrile illness study in Dhaka, Bangladesh. Healthy unexposed subjects recruited from Oxford gave written informed consent, and a Research Ethics Committee (16/YH/0247) approved the work. Written informed consent was obtained for all participants enrolled in the study, including for storage and export of samples to the UK. Studies were conducted according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. Peripheral blood mononuclear cell isolation PBMCs were isolated from whole blood within three hours of blood draw by density gradient centrifugation using Lymphoprep (AxisShield, Oslo, Norway) and subsequently cryopreserved in heat-inactivated fetal calf serum (FCS) and 10% (v/v) dimethyl sulfoxide (DMSO). Cells were kept at -80°C for short-term storage and then transferred to liquid nitrogen for long-term storage. Cells were thawed at 37 0 C and washed in R10 media composed of RPMI 1640 (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated FCS (Life Technologies, Carlsbad, CA, USA), 1% L-glutamine, and 1% penicillin/streptomycin (Sigma-Aldrich, Dorset, UK). Samples were treated with 1µl/ml benzonase (Merck Millipore, Billerica, MA, USA) in R10 for 30min in a 37⁰C, 5% CO2, and 95% humidity incubator and subsequently centrifuged and resuspended in R10. Cells were counted using a haemocytometer with trypan blue staining (Sigma) to assess viability before use in downstream assays. THP-1 cell maintenance and propagation A monocyte cell line was used as antigen presenting cell (APC) in all functional MAIT cell experiments. THP-1 cells (ECACC, UK) were cultured in RPMI 1640 with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin, and 0.05% β- mercaptoethanol (all Sigma-Aldrich, UK) at 2 x 10 5 cells/ml. Preparation of fixed bacteria Bacterial strains of E.coli (DH5-α), S. Typhi (PF CT18) and K. pneumoniae (ATCC 13368) were fixed with 2% paraformaldehyde (PFA) and B. pseudomallei (K96243) was fixed with 0.5% PFA before use in MAIT cytokine secretion assays. Bacteria were cultured in LB agar except for B. pseudomallei which was cultured using TSB and Ashdown’s medium. Colony forming units (CFU)/ml were determined by plating serial dilutions of bacteria prior to fixation. MAIT cytokine secretion assay (THP-1 co-culture) THP-1 cells were plated at a density of 80,000 cells per well in a 96-well round-bottom plate and stimulated with PFA-fixed BP, E. coli, S. Typhi and K. pneumoniae . Bacteria were used at a bacteria per cell ratio (BpC) of 30 for E. coli and 50 for the other bacteria unless otherwise stated in the figure legends. Staphylococcal enterotoxin B (SEB, final concentration: 5ug/ml ) was used as a positive control in all the experiments. PBMC were added to THP-1 cells at 80,000 cells/well and cultured overnight (16 hours) at 37°C, 5% CO2 and 95% humidity incubator. Brefeldin A (1:1000 final dilution; 20µl/well) was added to each well for the last 4 hours of incubation. Cells were subsequently stained for flow cytometry analysis. Cytokine secretion by THP-1 cells THP-1 cells were cultured with fixed E. coli and B. pseudomallei as above but in the absence of PBMC. Supernatants were collected after 20 hours and stored at -70ºC until further use. IL-12p40 was measured in cell-free supernatants (diluted 2-fold) using the IL-12p40 Duoset enzyme linked immunosorbent assay (ELISA) Kit according to manufacturer’s instructions (R&D Systems). Results were obtained as absorbance values (450nm) using a GloMax Explorer microplate reader (Promega). Concentrations of cytokines were calculated from standard curves using GraphPad Prism Version 10 (San Diego, CA, USA). Flow cytometry staining For activation and phenotyping experiments, 1x10 6 PBMC were re-suspended in phosphate buffered saline (PBS) and incubated for 20 minutes at 4⁰C with near-infrared live/dead fixable stain (Invitrogen, Carlsbad, CA, USA, 1/500) and fluorochrome-conjugated antibodies against extracellular markers: CD3, CD4, CD8, CD161, TCR Vα7.2, CD69 in the presence of human FcR blocking reagent (Miltenyi Biotec). After washing with PBS, cells were fixed with fixation/permeabilization solution (BD Biosciences, eBioscience) for 15 min at 4⁰C, washed with permeabilization buffer (BD Biosciences, eBioscience) followed by incubation with a fluorochrome-conjugated antibody against granzyme B (GzmB) in the presence of FcR blocking reagent for 30 minutes at 4⁰C. After washing with permeabilization buffer, cells were resuspended in PBS and stored at 4⁰C until acquisition. For the MAIT cytokine secretion assay, a one-step staining was performed. PBMC (1x10 6 ) were first incubated with viability dye as described above. After washing in PBS, cells were fixed with 2% PFA for 20min at 4⁰C, then stained with fluorochrome conjugated antibodies: CD3, CD4, CD8, CD161, TCR Vα7.2, IFN-γ, and TNF in permeabilization buffer for 20 minutes at 4⁰C. Finally, cells were washed with permeabilization buffer, resuspended in PBS and acquired on a MACSQuant Analyser 10 (Miltenyi Biotec). The acquisition was performed on the same day for all experiments. The following rules were used for quality control of flow cytometry data: a minimum of 1000 events in the CD3 gate and for downstream analysis of MAIT cells (CD69, GzmB, IFN-γ) a minimum of 50 events in the parent gate. Data analysis was performed with FlowJo Version 10 (FlowJo LLC, Ashland, OR, USA). Details of antibody clones, manufacturer and fluorochrome can be found in the Supplementary information Table 1 . Staining for TNF was performed in the assay but data are not presented in this study. Statistical analysis Categorical variables were expressed as counts and frequencies and compared using Fisher’s exact test. Continuous variables were expressed as mean and range for parametric data (age) and differences between groups were calculated using Student’s t -test. Non-parametric variables (immunological parameters, HbA1c) were expressed as median and interquartile range. For continuous variables, differences between two independent groups were calculated using the Mann-Whitney U test and differences between three independent groups were assessed using Kruskal Wallis test using Dunn’s multiple comparison. For comparisons of paired data Wilcoxon signed rank test was used to compare two groups and Friedman test followed by Dunn’s multiple comparison was used to compare three or more groups. A two-tailed p value of < 0.05 was considered statistically significant. To evaluate the association of continuous experimental variables with demographic and clinical characteristics we used a generalized linear model (GLM) with gamma distribution and log link. Statistical analysis was performed using SPSS Version 29 (IBM) and graphs were generated using GraphPad Prism Version 10. In all analyses, the statistical significance of the result is indicated with p values. Data availability Datasets generated during the current study are available from the corresponding author on reasonable request. Declarations Conflict of Interest Statement The authors declare no conflicts of interest. Funding Statement This work was supported by a Wellcome Trust Intermediate Clinical Fellowship award (WT100174/Z/12/Z) and an NIHR Global Research Professorship (NIHR300791) to SJD. FRC was supported by a Commonwealth Scholarship (BDCS-2015-44), UK. This research was funded in part, by the Wellcome Trust (220211/Z/20/Z). Supported by NIHR Biomedical Research Centre, Oxford. The views expressed are those of the author/s and not necessarily those of the NHS, the NIHR or the Department of Health. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. Author Contribution Conceptualization SJD, PK, CBW, BK, FRCMethodology CBW, AK, HM, PR, FRC, JH, BKCollection of Clinical Samples FRC, LB, MRA, MRH, PT, SJDFormal Analysis BK, FRCInvestigation FRC, SM, HAResources SJD, PKData Curation BK, FRCWriting – Original Draft BK, MZ, FRCWriting – Review & Editing FRC, MZ, PA, SM, HA, JH, MRA, LB, MRH, AK, HM, PR, DL, NPD, PC, PK, CBW, BK, SJDVisualization BK, PASupervision SJD, PK, CBW, BKFunding Acquisition SJD, FRC Acknowledgement We would like to thank all the study participants, their families as well as the staff at Sunpasitthiprasong Hospital, Ubon Ratchatani. 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Immunol. 13 , 862–874 (2013). Zheng, Y. et al. MAIT cell activation and recruitment in inflammation and tissue damage in acute appendicitis. Sci Adv 10 , (2024). Dias, J. et al. The CD4-CD8- MAIT cell subpopulation is a functionally distinct subset developmentally related to the main CD8 + MAIT cell pool. Proceedings of the National Academy of Sciences 115, (2018). Wong, E. B., Ndung’u, T. & Kasprowicz, V. O. The role of mucosal-associated invariant T cells in infectious diseases. Immunology 150 , 45–54 (2017). Dunachie, S. & Chamnan, P. The double burden of diabetes and global infection in low and middle-income countries. Trans. R Soc. Trop. Med. Hyg. 113 , 56–64 (2019). Magalhaes, I. et al. Mucosal-associated invariant T cell alterations in obese and type 2 diabetic patients. J. Clin. Invest. 125 , 1752–1762 (2015). Carolan, E. et al. Altered Distribution and Increased IL-17 Production by Mucosal-Associated Invariant T Cells in Adult and Childhood Obesity. J. 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Supplementary Files ChowdhuryetalMAITMelioidosisSupportingInformation2026.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 08 May, 2026 Reviews received at journal 08 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviews received at journal 15 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Editor invited by journal 18 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 16 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Susanna","middleName":"J","lastName":"Dunachie","suffix":""}],"badges":[],"createdAt":"2026-03-10 11:53:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9083519/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9083519/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107480616,"identity":"32aa5a7a-e961-4ba3-8b04-ebe7baed3b27","added_by":"auto","created_at":"2026-04-22 02:12:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":154260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eB. pseudomallei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activates MAIT cells in a cytokine dependent manner.\u003c/strong\u003e (A) Schematic representation of experimental setup. A MAIT cell activation assay was performed by culturing human peripheral blood mononuclear cells (PBMC), THP-1 cells and paraformaldehyde-fixed bacteria together for for 20 hours followed by intracellular cytokine staining by flow cytometry. BFA= brefeldin A. \u0026nbsp;(B) Gating strategy for identification of MAIT cells: within the CD3+ live cell gate different T cell populations were identified based on CD4 and CD8 expression. Subsequent gating for CD161\u003csup\u003ehi\u003c/sup\u003e and Vα 7.2+ identifies MAIT cells in the respective T cell populations. (C) Proportion of CD8+ MAIT cells expressing IFN-γ following co-culture with THP-1 cells and fixed \u003cem\u003eB. pseudomallei\u003c/em\u003e at different bacteria per cell ratio (BpC, n=3 healthy individuals). (D) Paired analysis of IFN-γ expression upon culture with THP-1 cells and fixed bacteria (\u003cem\u003eB. pseudomallei\u003c/em\u003e 50 BpC; \u003cem\u003eE. coli\u003c/em\u003e 30 BpC) compared to respective media control (n=5 healthy individuals). (E) Blocking experiment assessing the role of cytokines and TCR activation (n=5 healthy individuals). MAIT cell induced IFN-γ expression was measured upon stimulation with THP-1 cells and fixed \u003cem\u003eB. pseudomallei \u003c/em\u003e(50 BpC) in the presence or absence of blocking antibodies against MR-1, IL-12, IL-18 and an isotype control. A two-tailed p value of \u0026lt;0.05 was considered statistically significant. Exact p values \u0026lt;0.1 are displayed on graphs. The data are displayed as spaghetti plots or box and whiskers plot. Paired comparisons were analysed by Wilcoxon rank test for two groups and Friedman test followed by Dunn’s multiple comparison for greater than two groups.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9083519/v1/395e7cd7a1ff421622a0d0a9.jpg"},{"id":107043110,"identity":"268f60bd-9577-4ea9-8fc7-f9755bba3865","added_by":"auto","created_at":"2026-04-16 06:46:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":210775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirculating MAIT cells are reduced, activated and dysfunctional in acute melioidosis.\u003c/strong\u003e (A) Schematic representation of study design. PBMC from acute and recovered melioidosis patients alongside healthy endemic controls (uninfected) were analysed by flow cytometry for MAIT cell frequency, activation (CD69) and cytotoxic granules (GzmB) in MAIT cells \u003cem\u003eex vivo\u003c/em\u003e as well as \u003cem\u003ein vitro\u003c/em\u003eIFN-γ expression following stimulation with fixed bacteria. (B) Frequency of MAIT cells within the CD8+ and DN T-cell population, (C) expression of CD69 and GzmB in CD8+ and DN MAIT cells and (D) expression of IFN-γ in MAIT cells upon \u003cem\u003ein vitro\u003c/em\u003e stimulation with fixed \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eB. pseudomallei\u003c/em\u003e (BP), staphylococcal enterotoxin B (SEB) and media were compared between controls, acute melioidosis patients and those who had recovered from melioidosis (1 year post admission). Grey circles: controls, orange squares: acute patients, brown triangles: recovered patients. The number of biological replicates per group is given in brackets under graphs. A two-tailed p value of \u0026lt;0.05 was considered statistically significant. Exact p values \u0026lt;0.1 are displayed on graphs. The data are displayed as median and interquartile range. The three groups were compared using Kruskal Wallis test followed by Dunn’s multiple comparison. DM= diabetes mellitus.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9083519/v1/3e7754adb4f3093dbd308ef8.jpg"},{"id":107043111,"identity":"c7e3a681-782f-48ca-a83f-3108c65c112d","added_by":"auto","created_at":"2026-04-16 06:46:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":166404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional changes were observed in circulating MAIT cells in acute melioidosis survivors compared to those who died.\u003c/strong\u003e(A) Frequency of MAIT cells within the CD8+ and DN T-cell population, (B) expression of CD69 and GzmB in CD8+ and DN MAIT cells and (C) expression of IFN-γ in MAIT cells following \u003cem\u003ein vitro\u003c/em\u003e stimulation with fixed \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eB. pseudomallei\u003c/em\u003e (BP), staphylococcal enterotoxin B (SEB) and media were compared between acute melioidosis patients who died (red squares) within 28 days of admission and those who survived (grey circles). The number of biological replicates per group is given in brackets under graphs. A two-tailed p value of \u0026lt;0.05 was considered statistically significant. Exact p values \u0026lt;0.1 are displayed on graphs. The data are displayed as median and interquartile range. The two groups were compared using Mann Whitney U test.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9083519/v1/802130ce84f6783f32b108e1.jpg"},{"id":107480625,"identity":"5aced2d2-5030-4c50-9c4c-4552ca9a1295","added_by":"auto","created_at":"2026-04-22 02:12:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiabetes drives MAIT cell changes in acute melioidosis.\u003c/strong\u003e The acute and recovered melioidosis cohorts were split according to diabetes mellitus (DM) status. (A, D) The frequency of MAIT cells within the CD8+ and DN T-cell population, (B, E) expression of CD69 and GzmB in CD8+ and DN MAIT cells and (C, F) expression of IFN-γin MAIT cells upon \u003cem\u003ein vitro\u003c/em\u003e stimulation with fixed \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eB. pseudomallei\u003c/em\u003e (BP), staphylococcal enterotoxin B (SEB) and media were compared between the non-DM (circles) and DM (squares) group. The number of biological replicates per group is given in brackets under graphs. A two-tailed p value of \u0026lt;0.05 was considered statistically significant. Exact p values \u0026lt;0.1 are displayed on graphs. The data are displayed as median and interquartile range. The two groups were compared using Mann Whitney U test.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9083519/v1/d03ce07590e5803b454491b1.jpg"},{"id":109067441,"identity":"3c5513a5-44b2-468c-854b-43ae13fd8e11","added_by":"auto","created_at":"2026-05-12 09:51:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1161795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9083519/v1/4fe75851-5029-462c-916e-794f375604ae.pdf"},{"id":107043108,"identity":"6eaeb61b-f51e-4185-b63b-3debd70b2632","added_by":"auto","created_at":"2026-04-16 06:46:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":334041,"visible":true,"origin":"","legend":"","description":"","filename":"ChowdhuryetalMAITMelioidosisSupportingInformation2026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9083519/v1/3794fc00216ec4e4745f55a4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mucosal-associated invariant T (MAIT) cells are reduced and dysfunctional in acute melioidosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e resulting in millions of deaths each year\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Bacterial infections are responsible for a major proportion of sepsis cases in the world, and among them Gram-negative bacteria are the main culprit\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Some of the commonest causative Gram-negative organisms are, \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae, Pseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Typhi\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In addition, the Gram-negative bacterium \u003cem\u003eBurkholderia pseudomallei\u003c/em\u003e (BP) which causes the neglected tropical disease melioidosis is an important cause of adult sepsis in Asia\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. With an estimated incidence of 165,000 annual cases worldwide\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and an in-hospital case fatality rate of up to 50% in some regions of Thailand\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, melioidosis causes a significant burden in endemic regions. Several risk factors including diabetes mellitus (DM), chronic kidney and lung disease, malignancy and excessive alcohol use are associated with melioidosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. DM constitutes the biggest risk factor with a 12-fold increased risk of melioidosis, and over 50% of melioidosis patients have this metabolic disease\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We and others have previously demonstrated a role for T cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and natural killer (NK) cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e in survival from melioidosis in humans, as well as differences in immunological pathways associated with survival in patients with DM co-morbidity\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Recent studies in mouse models point towards an important contribution of unconventional T cells to protection. In the absence of invariant NKT (iNKT) cells, survival time was reduced in \u003cem\u003eB. pseudomallei\u003c/em\u003e infected BALB/c mice, and early activation of this cell subset contributed to bacterial clearance and enhanced survival\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In a C57B6/J mouse model of pulmonary melioidosis the presence of γδT cells was important for survival via a reduction of neutrophil-induced inflammation in the lungs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMucosal-associated invariant T (MAIT) cells are another unconventional T cell subset with both innate and adaptive properties\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This cell population plays an important role in human immune responses to bacterial and viral infections\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. MAIT cells are abundant in the gut, liver, lung and blood, comprising 1\u0026ndash;10% of peripheral blood T lymphocytes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In humans, MAIT cells express a conserved invariant T cell receptor (TCR) α-chain (Vα7.2-Jα33) and are defined by their restriction to the non-classical MHC class I-related (MR-1) molecule\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Their natural ligands are derived from folic acid (vitamin B9) and from an intermediate in the microbial biosynthesis of riboflavin (vitamin B2)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, organisms which synthesise riboflavin precursors (Gram-negative bacteria, Yeasts) can stimulate MAIT cells in an MR-1-dependent manner\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. MAIT cells can also be activated by cytokines in a TCR-independent manner. Notably, IL-12 and IL-18 are potent inducers of MAIT effector functions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Established mouse models demonstrate a beneficial role of MAIT cells in immune defence against several bacterial pathogens including \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eFrancisella tularensis\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eLegionella longbeachae\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and extraintestinal pathogenic \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In humans with severe sepsis and septic shock, an early reduction of MAIT cell numbers can be observed compared to healthy controls \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The role of MAIT cells in melioidosis has not been explored before. In this study we characterise the ability of BP to activate MAIT cells and how MAIT cell frequency and function is affected in a cohort of acute melioidosis patients from Thailand. We further assess the contribution of DM co-morbidity on the properties of these important effector cells.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eB. pseudomallei activates MAIT cells in vitro in a cytokine dependent manner\u003c/h2\u003e \u003cp\u003eWe first assessed the ability of BP to induce IFN-γ secretion from MAIT cells in a previously established co-culture assay\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e using healthy PBMC and THP-1 cells incubated with PFA-fixed BP at 25, 50 and 75 bacteria per cell (BpC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). MAIT cells were phenotypically characterised by the expression of Vα7.2 and high expression of CD161 in T cell subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For this analysis we focused on MAIT cells within the CD8\u0026thinsp;+\u0026thinsp;T cell subset as this constitutes the majority of MAIT cells in human blood\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. BP induced IFN-γ secretion from circulating CD8\u0026thinsp;+\u0026thinsp;MAIT cells, with the strongest induction cytokine expression at BpC 50 (50 BP per THP1 cell). Incubation with 50 BP per THP1 cell was used in all subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In comparison to \u003cem\u003eE. coli\u003c/em\u003e, used at the previously optimised dose of BpC 30\u003csup\u003e25\u003c/sup\u003e, BP induced three-fold lower levels of IFN-γ expression in CD8\u0026thinsp;+\u0026thinsp;MAIT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, secretion of IL-12 by THP-1 cells in response to fixed \u003cem\u003eE.coli\u003c/em\u003e was 3-fold higher compared to that induced by fixed BP (\u003cem\u003eE.coli\u003c/em\u003e: 2500 pg/ml, BP: 782 pg/ml). Blocking of both IL-12 and IL-18 during the incubation of BP with THP-1 cells completely abrogated IFN-γ expression (median: 1.4, IQR: 0.5\u0026ndash;2.4% IFN-γ positive) compared to the isotype control (median: 10.7, IQR: 5.3\u0026ndash;11.1% IFN-γ positive, p\u0026thinsp;=\u0026thinsp;0.0149), indicating that IFN-γ production was predominantly cytokine-dependent. Blocking of MR-1 only marginally reduced cytokine production, and there was no synergistic effect of blocking both cytokines and MR-1 in this culture system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This demonstrates that BP is able to activate MAIT cells in a predominantly cytokine-dependent manner at the assessed timepoint.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCirculating MAIT cells are reduced and dysfunctional in acute melioidosis\u003c/h3\u003e\n\u003cp\u003eHaving demonstrated that MAIT cells are activated by BP \u003cem\u003ein vitro\u003c/em\u003e we next sought to study the frequency, activation and function of MAIT cells \u003cem\u003eex vivo\u003c/em\u003e in the context of acute melioidosis infection. We used samples from a cohort of acute and recovered melioidosis patients recruited in Thailand between 2012\u0026ndash;2017. Individuals with and without DM from the same region were used as endemic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Acute melioidosis patients were slightly but significantly older (median 59, IQR 51\u0026ndash;65 years, p\u0026thinsp;=\u0026thinsp;0.016) than recovered patients (median 53, IQR 45\u0026ndash;61 years) and healthy controls (median 52, IQR 48\u0026ndash;61 years). There were no differences in sex, the proportion of individuals with DM, or history of renal impairment between groups. We found a 2.7-fold reduction in frequency of circulating MAIT cells within the double negative (DN) T cell compartment in acute melioidosis cases (median 2.8, IQR 0.9\u0026ndash;4.7%) compared with healthy controls (median 7.5, IQR 2.0-11.8%, p\u0026thinsp;=\u0026thinsp;0.0059) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e Importantly, one year after infection the frequency of DN MAIT cells in recovered patients (median 6.7, IQR 4.2\u0026ndash;11.3%) returned to levels comparable to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The same effect was observed when comparing the frequency of MAIT cells in the overall live cell compartment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). In contrast, the frequency of CD8\u0026thinsp;+\u0026thinsp;MAIT cells remained unaffected during acute disease.\u003c/p\u003e \u003cp\u003eNext, we assessed activation and functional properties of MAIT cells. Due to the low numbers of MAIT cells in acute disease we combined CD8\u0026thinsp;+\u0026thinsp;and DN MAIT cell compartments in order to increase events for detection of downstream parameters by flow cytometry. MAIT cells (CD8\u0026thinsp;+\u0026thinsp;and DN combined) were highly activated (CD69+) in acute melioidosis (median 49.1, IQR 35.5\u0026ndash;60.1%) compared with healthy controls (median 11.6, IQR 3.7\u0026ndash;23.2%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and recovered patients (median 4.7, IQR 2.5\u0026ndash;15.4%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). MAIT cells displayed higher GzmB expression in acute disease (median 8.6, IQR 5.5\u0026ndash;27.5%) compared with healthy controls, (median 3.3, IQR 1.7\u0026ndash;15.8%) although this difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.0979) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, IFN-γ production by MAIT cells from acute melioidosis patients following \u003cem\u003ein vitro\u003c/em\u003e stimulation was abrogated compared with healthy controls. Responses to BP were significantly reduced (median, IQR: Acute 0.4, 0-1.2%; Healthy 6.1, 1.6\u0026ndash;13.5%, p\u0026thinsp;=\u0026thinsp;0.0006) and albeit not reaching statistical significance we also noted lower reactivity to \u003cem\u003eE. coli\u003c/em\u003e (median, IQR: Acute 1.0, 0.2\u0026ndash;8.4%; Healthy 11.0, 3.6\u0026ndash;19.4%, p\u0026thinsp;=\u0026thinsp;0.0580). Upon recovery, IFN- γ expression in response to BP returned to levels comparable with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). To assess whether a reduced capacity for IFN-γ expression in MAIT cells is a particular feature of melioidosis or a more common effect in sepsis we measured MAIT cell IFN-γ expression in a small cohort of sepsis patients with different disease aetiology in Bangladesh. MAIT cell IFN-γ expression upon stimulation with the respective causative pathogen was significantly impaired in patients infected with \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;3B, C\u003c/b\u003e) and a trend for reduced levels was observed in those infected with \u003cem\u003eS.\u003c/em\u003e Typhi (\u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e) compared with healthy controls. Overall, our data highlights depletion of MAIT cells in the DN T cell compartment in acute melioidosis, and shows that despite being highly activated in acute disease, MAIT cells are limited in their ability to produce IFN-γ in response to bacterial stimulation \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticipant characteristics for melioidosis cases (acute and recovered) and endemic controls.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMelioidosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcute\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRecovered\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;55\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;24\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;41\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian age\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33\u0026ndash;84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u0026ndash;88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33\u0026ndash;73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInterquartile range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51\u0026ndash;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45\u0026ndash;61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48\u0026ndash;61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17 (41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 (71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (59%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003e28-Day Mortality, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvived\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDied\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCo-Morbidities, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiabetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31 (56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (54%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistory of renal impairment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (21%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (NA%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Pearson Chi-Squared test; Kruskal-Wallis rank sum test; Fisher's exact test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImpaired GzmB and IFN-γ expression in MAIT cells of non-survivors\u003c/h3\u003e\n\u003cp\u003eWe have previously shown that IFN-γ secretion by conventional T cells is impaired in acute melioidosis patients who died compared to those who survived\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To address whether a similar effect is observed for MAIT cells we stratified the acute melioidosis cohort based on clinical outcome (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e). While frequency and activation (CD69+) of MAIT cells were comparable in patients who survived and those who died within 28-days of hospital admission (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e), GzmB expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and IFN-γ production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) in response to BP were reduced in non-survivors (GzmB median, IQR: Survived 19.5, 6.3\u0026ndash;29.2%; Died 7.3, 2.6\u0026ndash;12.2%, p\u0026thinsp;=\u0026thinsp;0.0564; IFN-γ median, IQR: Survived 0.7, 0.3\u0026ndash;1.5%; Died 0.0, 0.0-1.2%, p\u0026thinsp;=\u0026thinsp;0.0404). This shows a further impairment of MAIT cell function in patients with fatal outcome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMAIT cell frequency and function are reduced in acute melioidosis patients with DM\u003c/h3\u003e\n\u003cp\u003eSince DM is an independent risk factor for melioidosis, we compared the frequency and activation of MAIT cells in acute and recovered melioidosis patients between those with and without DM (\u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e). Acute melioidosis patients with DM showed a 50% reduction in the proportion of MAIT cells within the DN T cell compartment (median 2.1, IQR 0.6\u0026ndash;3.2%) compared with the non-DM group (median 4.4, IQR 2.0-7.3%, p\u0026thinsp;=\u0026thinsp;0.0136). No difference was observed for the CD8\u0026thinsp;+\u0026thinsp;MAIT cell subset (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). MAIT cell expression of CD69 and GzmB was not significantly different in acute patients with DM compared to those without but we note that GzmB expression is slightly elevated in the DM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Importantly, IFN-γ production by MAIT cells in response to \u003cem\u003eE. coli\u003c/em\u003e was significantly impaired in acute melioidosis patients with DM (median 0.3, IQR 0.0-2.4%) compared to those without DM (median 5.1, IQR 1.0-11.2%, p\u0026thinsp;=\u0026thinsp;0.0181, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In contrast. recovered patients did not show differences in cell frequency, activation or function based on DM status (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F) indicating that MAIT cell dysfunction in DM is context dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAssociation of MAIT cell features with clinical parameters\u003c/h3\u003e\n\u003cp\u003eIn order to assess the combined impact of clinical and demographic factors on MAIT cell frequency and activation we performed GLM analysis using age, sex, DM status, 28-day mortality and history of renal impairment as covariates. In line with univariate analysis, we identified DM as being significantly associated with a reduced proportion of MAIT cells within the DN T cell compartment and 28-day mortality being associated with lower expression of GzmB in MAIT cells (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, DM was independently associated with significantly higher expression of GzmB in MAIT cells when taking relevant covariates into account. Differences in CD69 expression on MAIT cells were not associated with any of the tested variables.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneralized linear model testing the association of MAIT frequency and activation with demographic and clinical parameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eEstimates\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDependent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePredictor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSE\u003csub\u003eCoef\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eDN MAIT % of T cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.672\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28-day Mortality (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.098\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiabetes (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-0.566\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.047\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistory of renal impairment (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.084\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eCD69% of MAIT cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28-day Mortality (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiabetes (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.313\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistory of renal impairment (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.265\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eGzmB % of MAIT cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.971\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.629\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28-day Mortality (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-0.854\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.037\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiabetes (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.070\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.012\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistory of renal impairment (yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.413\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eGamma log linked, Coef=Coefficient, SE=standard error\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis is the first study to characterise the MAIT cell response in the context of the neglected tropical disease melioidosis. Here we show that BP efficiently induces IFN-γ secretion by Vα7.2\u0026thinsp;+\u0026thinsp;CD161hi MAIT cells in an overnight (20 hours) co-culture system with THP-1 cells. In our hands, blocking of cytokine signalling using antibodies targeted at the receptors for IL-12 and IL-18 completely abrogated the expression of IFN-γ in MAIT cells with no further reduction achieved by additional blocking of MR-1. Ussher et al\u003csup\u003e30\u003c/sup\u003e previously demonstrated a time dependent increase in IFN-γ production by MAIT cells stimulated with \u003cem\u003eE. coli\u003c/em\u003e, with early IFN-γ secretion (5 hours) being entirely dependent on MR-1, and late IFN-γ secretion (20 hours) dependent on MR-1 in combination with IL-12 and IL-18. Similarly, \u003cem\u003eC. difficile\u003c/em\u003e-induced IFN-γ production was found to be initially MR-1 dependent, with a more pronounced role for cytokines in driving IFN-γ production and cytotoxic features after longer incubation times\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In contrast, our data does not provide evidence for combined action of MR-1 and cytokines in induction of MAIT cell IFN-γ in response to BP after overnight culture, suggesting predominantly TCR-independent activation. The extent to which MAIT cells secrete IFN-γ is dependent on the levels of MR-1 upregulation, TLR stimulation and induction of IL-12 in antigen presenting cells (APC)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eE. faecalis\u003c/em\u003e, which is a weak inducer of IL-12 in THP-1 cells, also induced weaker MAIT IFN-γ responses compared to \u003cem\u003eE coli\u003c/em\u003e, suggesting that MAIT cell activation and cytokine release are pathogen-specific\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In line with this, we show that BP compared with \u003cem\u003eE. coli\u003c/em\u003e is a weaker inducer of IL-12 secretion by primed THP-1 cells, likely resulting in lower induction of IFN-γ by MAIT cells.\u003c/p\u003e \u003cp\u003eIn a cohort of melioidosis patients in Thailand, we show that MAIT cell frequency is significantly reduced during acute infection, with the cells that are present being highly activated but dysfunctional. We observed dampened IFN-γ production by MAIT cells in response to BP and \u003cem\u003eE.coli\u003c/em\u003e, with responses returning to normal upon recovery. Within the acute melioidosis group we did not find evidence for differences in MAIT cell frequency and activation based on outcome of infection, but impaired function including reduced GzmB expression and IFN-γ production was noted in patients who died within 28-days of hospital admission.\u003c/p\u003e \u003cp\u003eOur findings align with other studies of MAIT cells in acute bacterial disease. Marked specific depletion of MAIT cells was found in acute non-streptococcal septic patients compared to healthy controls, and this was associated with susceptibility to nosocomial infection\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Similar findings have been found in inflammatory and infectious diseases including acute cholecystitis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, pulmonary tuberculosis (TB)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and scrub typhus\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In a recent \u003cem\u003eS\u003c/em\u003e. Typhi human challenge study, CD8\u0026thinsp;+\u0026thinsp;MAIT cell frequency was sharply decreased early post-infection in volunteers who developed typhoid disease compared to those resistant to disease\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Reduced circulating MAIT numbers in infection could be due to recruitment of the cells into sites of infection, or increased apoptosis in the case of sepsis. As demonstrated and reviewed by Hotchkiss et al\u003csup\u003e40\u003c/sup\u003e, extensive apoptosis of immune cells including T cells, B cells and dendritic cells is a hallmark of sepsis. Zheng et al have recently shown that MAIT cells are reduced in circulation but enriched in tissue during acute appendicitis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Interestingly, the reduction in MAIT cells in acute melioidosis was specific to the DN T cell compartment and not the CD8\u0026thinsp;+\u0026thinsp;T cell subset. Notably, DN MAIT cells have been shown to exhibit higher apoptotic gene signatures and they are more prone to activation-induced apoptosis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e possibly explaining the cell subset specific depletion observed in our study. In acute bacterial infection, there is evidence of diminished functional capacity of circulating MAIT cells\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. MAIT cell IFN-γ production has been previously shown to be reduced during active mycobacterial infection\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, which is in line with our findings.\u003c/p\u003e \u003cp\u003eWe identified specific changes in MAIT cells during acute melioidosis in patients with DM, including reduced proportion of DN MAIT cells, increased GzmB expression and reduced IFN-γ production upon stimulation with bacteria. DM, the major risk factor for melioidosis, is associated with chronic inflammation, reduced T cell function, and dysfunction of the innate immune system; specifically neutrophil and monocyte function\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. MAIT cells have been previously shown to be reduced in frequency and function in blood and adipose tissue of Type 2 DM (T2DM) patients and obese individuals\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Magalhaes et al have shown that MAIT cells from T2DM patients have a pro-inflammatory phenotype characterised by higher levels of IL-17, IL-2, IFN-γ and GzmB compared to healthy individuals when activated with PMA/Ionomycin \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In contrast, MAIT cells were less activated upon specific TCR activation and showed a skewed cytokine response away from IFN-γ and TNF towards an IL-17 biased response in T2DM compared to healthy controls\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. A subset of IL-17 secreting MAIT cells has also been associated with various chronic inflammatory conditions\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Our study shows similar impairment of MAIT cell function in the context of acute bacterial infection with reduced IFN-γ secretion in response to \u003cem\u003eE. coli\u003c/em\u003e in patients DM co-morbidity. Although beyond the scope of this study, it will be interesting to follow up the IL-17 bias in the context of melioidosis and DM. Overall, our results point toward specific functional alterations of MAIT cells in people with DM during acute melioidosis infection. MAIT cell function was generally low in acute melioidosis with a further reduction seen in those who died from the disease. Future studies are needed to specifically address the contribution of MAIT cells to disease susceptibility in people with DM and their role in early control of bacterial infections such as melioidosis.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003eThe number of MAIT cells was highly reduced in acute disease therefore analysis of downstream properties of MAIT cells was restricted to samples with enough cells. We characterised MAIT cells by their expression of CD161 and Vα7.2 and did not use 5-OP-RU loaded MR1 tetramers for a more direct identification of MAIT cells. We focussed on the measurement of IFN-γ as the main effector molecule produced by MAIT cells upon activation. Measuring IL-17 especially in the context of DM co-morbidity and local immune responses in the lungs might give further insights into the role of MAIT cells in disease susceptibility and melioidosis pathogenicity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePatient recruitment\u003c/h2\u003e \u003cp\u003eWe used a patient cohort recruited into an observational study of acute melioidosis at Sunpasitthiprasong Hospital, Ubon Ratchathani, Thailand between 2012-2017\u003csup\u003e11,12\u003c/sup\u003e. Patients over 18 years with culture-confirmed melioidosis at a median of 5 days after admission to hospital (IQR 4\u0026ndash;6 days)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e were included in the study. 67% of patients had a diagnosis of DM (defined for the purpose of this study as a past medical history of diabetes and/or a blood glycated haemoglobin (HbA1c) of \u0026ge;\u0026thinsp;7%)\u003csup\u003e12,48\u003c/sup\u003e. 28-day mortality was determined using hospital mortality records and follow-up telephone calls. Recovered melioidosis patients were seen one year after initial enrolment (week 52). Uninfected individuals with and without DM were recruited as endemic controls. Gram-negative culture-confirmed sepsis cases were recruited as part of the \u003cem\u003eAetiology of Fever\u003c/em\u003e study at Dhaka Medical College Hospital (DMCH) and Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM) Hospitals, Dhaka, Bangladesh. Adult patients of 18 years or more who were admitted with a history of fever\u0026thinsp;\u0026ge;\u0026thinsp;38 degrees for more than 48 hours were included. Acute Gram-negative sepsis patients (defined according to SIRS criteria)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e were admitted 1\u0026ndash;2 weeks into the course of their illness. Healthy endemic control subjects were recruited from the blood transfusion department of DMCH, and critically ill non-infectious patients (acute stroke, acute pesticide poisoning, diabetic ketoacidosis, severe acute asthma and acute exacerbation of COPD) were enrolled from the medicine department at DMCH. Healthy controls from the UK were recruited as part of the GI Biobank study at the University of Oxford and samples were used for MAIT \u003cem\u003ein vitro\u003c/em\u003e assays including blocking experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthical approvals\u003c/h2\u003e \u003cp\u003e The ethics committees of the Faculty of Tropical Medicine, Mahidol University (TMEC-12-014 and TMEC-15-046), of Sunpasitthiprasong Hospital, Ubon Ratchathani (018/2555 and 017/2559) and the Oxford Tropical Medicine Research Ethics Committee (OXTREC; 64\u0026thinsp;\u0026minus;\u0026thinsp;11 and 35\u0026thinsp;\u0026minus;\u0026thinsp;15) approved the Thai melioidosis study protocol. The ethics committee of DMC (MEU-DMC/ECC/2015/97), BIRDEM Hospital (BADAS-ERC/EC/17/0127), and OXTREC (OXTREC: 51\u0026thinsp;\u0026minus;\u0026thinsp;16) approved aetiology of febrile illness study in Dhaka, Bangladesh. Healthy unexposed subjects recruited from Oxford gave written informed consent, and a Research Ethics Committee (16/YH/0247) approved the work. Written informed consent was obtained for all participants enrolled in the study, including for storage and export of samples to the UK. Studies were conducted according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePeripheral blood mononuclear cell isolation\u003c/h2\u003e \u003cp\u003ePBMCs were isolated from whole blood within three hours of blood draw by density gradient centrifugation using Lymphoprep (AxisShield, Oslo, Norway) and subsequently cryopreserved in heat-inactivated fetal calf serum (FCS) and 10% (v/v) dimethyl sulfoxide (DMSO). Cells were kept at -80\u0026deg;C for short-term storage and then transferred to liquid nitrogen for long-term storage. Cells were thawed at 37\u003csup\u003e0\u003c/sup\u003eC and washed in R10 media composed of RPMI 1640 (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated FCS (Life Technologies, Carlsbad, CA, USA), 1% L-glutamine, and 1% penicillin/streptomycin (Sigma-Aldrich, Dorset, UK). Samples were treated with 1\u0026micro;l/ml benzonase (Merck Millipore, Billerica, MA, USA) in R10 for 30min in a 37⁰C, 5% CO2, and 95% humidity incubator and subsequently centrifuged and resuspended in R10. Cells were counted using a haemocytometer with trypan blue staining (Sigma) to assess viability before use in downstream assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTHP-1 cell maintenance and propagation\u003c/h2\u003e \u003cp\u003eA monocyte cell line was used as antigen presenting cell (APC) in all functional MAIT cell experiments. THP-1 cells (ECACC, UK) were cultured in RPMI 1640 with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin, and 0.05% β- mercaptoethanol (all Sigma-Aldrich, UK) at 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of fixed bacteria\u003c/h2\u003e \u003cp\u003eBacterial strains of \u003cem\u003eE.coli\u003c/em\u003e (DH5-α), \u003cem\u003eS.\u003c/em\u003e Typhi (PF CT18) and \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 13368) were fixed with 2% paraformaldehyde (PFA) and \u003cem\u003eB. pseudomallei\u003c/em\u003e (K96243) was fixed with 0.5% PFA before use in MAIT cytokine secretion assays. Bacteria were cultured in LB agar except for \u003cem\u003eB. pseudomallei\u003c/em\u003e which was cultured using TSB and Ashdown\u0026rsquo;s medium. Colony forming units (CFU)/ml were determined by plating serial dilutions of bacteria prior to fixation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMAIT cytokine secretion assay (THP-1 co-culture)\u003c/h2\u003e \u003cp\u003eTHP-1 cells were plated at a density of 80,000 cells per well in a 96-well round-bottom plate and stimulated with PFA-fixed BP, \u003cem\u003eE. coli, S.\u003c/em\u003e Typhi and \u003cem\u003eK. pneumoniae\u003c/em\u003e. Bacteria were used at a bacteria per cell ratio (BpC) of 30 for \u003cem\u003eE. coli\u003c/em\u003e and 50 for the other bacteria unless otherwise stated in the figure legends. Staphylococcal enterotoxin B (SEB, final concentration: 5ug/ml ) was used as a positive control in all the experiments. PBMC were added to THP-1 cells at 80,000 cells/well and cultured overnight (16 hours) at 37\u0026deg;C, 5% CO2 and 95% humidity incubator. Brefeldin A (1:1000 final dilution; 20\u0026micro;l/well) was added to each well for the last 4 hours of incubation. Cells were subsequently stained for flow cytometry analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCytokine secretion by THP-1 cells\u003c/h2\u003e \u003cp\u003eTHP-1 cells were cultured with fixed \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. pseudomallei\u003c/em\u003e as above but in the absence of PBMC. Supernatants were collected after 20 hours and stored at -70\u0026ordm;C until further use. IL-12p40 was measured in cell-free supernatants (diluted 2-fold) using the IL-12p40 Duoset enzyme linked immunosorbent assay (ELISA) Kit according to manufacturer\u0026rsquo;s instructions (R\u0026amp;D Systems). Results were obtained as absorbance values (450nm) using a GloMax Explorer microplate reader (Promega). Concentrations of cytokines were calculated from standard curves using GraphPad Prism Version 10 (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry staining\u003c/h2\u003e \u003cp\u003eFor activation and phenotyping experiments, 1x10\u003csup\u003e6\u003c/sup\u003e PBMC were re-suspended in phosphate buffered saline (PBS) and incubated for 20 minutes at 4⁰C with near-infrared live/dead fixable stain (Invitrogen, Carlsbad, CA, USA, 1/500) and fluorochrome-conjugated antibodies against extracellular markers: CD3, CD4, CD8, CD161, TCR Vα7.2, CD69 in the presence of human FcR blocking reagent (Miltenyi Biotec). After washing with PBS, cells were fixed with fixation/permeabilization solution (BD Biosciences, eBioscience) for 15 min at 4⁰C, washed with permeabilization buffer (BD Biosciences, eBioscience) followed by incubation with a fluorochrome-conjugated antibody against granzyme B (GzmB) in the presence of FcR blocking reagent for 30 minutes at 4⁰C. After washing with permeabilization buffer, cells were resuspended in PBS and stored at 4⁰C until acquisition. For the MAIT cytokine secretion assay, a one-step staining was performed. PBMC (1x10\u003csup\u003e6\u003c/sup\u003e) were first incubated with viability dye as described above. After washing in PBS, cells were fixed with 2% PFA for 20min at 4⁰C, then stained with fluorochrome conjugated antibodies: CD3, CD4, CD8, CD161, TCR Vα7.2, IFN-γ, and TNF in permeabilization buffer for 20 minutes at 4⁰C. Finally, cells were washed with permeabilization buffer, resuspended in PBS and acquired on a MACSQuant Analyser 10 (Miltenyi Biotec). The \u003cb\u003eacquisition\u003c/b\u003e was performed on the same day for all experiments. The following rules were used for quality control of flow cytometry data: a minimum of 1000 events in the CD3 gate and for downstream analysis of MAIT cells (CD69, GzmB, IFN-γ) a minimum of 50 events in the parent gate. Data analysis was performed with FlowJo Version 10 (FlowJo LLC, Ashland, OR, USA). Details of antibody clones, manufacturer and fluorochrome can be found in the \u003cb\u003eSupplementary information\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Staining for TNF was performed in the assay but data are not presented in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCategorical variables were expressed as counts and frequencies and compared using Fisher\u0026rsquo;s exact test. Continuous variables were expressed as mean and range for parametric data (age) and differences between groups were calculated using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Non-parametric variables (immunological parameters, HbA1c) were expressed as median and interquartile range. For continuous variables, differences between two independent groups were calculated using the Mann-Whitney U test and differences between three independent groups were assessed using Kruskal Wallis test using Dunn\u0026rsquo;s multiple comparison. For comparisons of paired data Wilcoxon signed rank test was used to compare two groups and Friedman test followed by Dunn\u0026rsquo;s multiple comparison was used to compare three or more groups. A two-tailed p value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. To evaluate the association of continuous experimental variables with demographic and clinical characteristics we used a generalized linear model (GLM) with gamma distribution and log link. Statistical analysis was performed using SPSS Version 29 (IBM) and graphs were generated using GraphPad Prism Version 10. In all analyses, the statistical significance of the result is indicated with p values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eDatasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Statement\u003c/h2\u003e \u003cp\u003eThis work was supported by a Wellcome Trust Intermediate Clinical Fellowship award (WT100174/Z/12/Z) and an NIHR Global Research Professorship (NIHR300791) to SJD. FRC was supported by a Commonwealth Scholarship (BDCS-2015-44), UK. This research was funded in part, by the Wellcome Trust (220211/Z/20/Z). Supported by NIHR Biomedical Research Centre, Oxford. The views expressed are those of the author/s and not necessarily those of the NHS, the NIHR or the Department of Health. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization SJD, PK, CBW, BK, FRCMethodology CBW, AK, HM, PR, FRC, JH, BKCollection of Clinical Samples FRC, LB, MRA, MRH, PT, SJDFormal Analysis BK, FRCInvestigation FRC, SM, HAResources SJD, PKData Curation BK, FRCWriting \u0026ndash; Original Draft BK, MZ, FRCWriting \u0026ndash; Review \u0026amp; Editing FRC, MZ, PA, SM, HA, JH, MRA, LB, MRH, AK, HM, PR, DL, NPD, PC, PK, CBW, BK, SJDVisualization BK, PASupervision SJD, PK, CBW, BKFunding Acquisition SJD, FRC\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e We would like to thank all the study participants, their families as well as the staff at Sunpasitthiprasong Hospital, Ubon Ratchatani. We thank the Oxford University Hospitals NHS Foundation Trust microbiology laboratory for kindly providing K. pneumoniae and the Oxford Vaccine Group for providing S. Typhi for this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eDatasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShankar-Hari, M. et al. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock. \u003cem\u003eJAMA\u003c/em\u003e \u003cb\u003e315\u003c/b\u003e, 775 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaniels, R., Nutbeam, T. \u0026amp; Berry, E. 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Immunol.\u003c/em\u003e \u003cb\u003e194\u003c/b\u003e, 5775\u0026ndash;5780 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePisarska, M. M., Dunne, M. R., O\u0026rsquo;Shea, D. \u0026amp; Hogan, A. E. Interleukin-17 producing mucosal associated invariant T cells ‐ emerging players in chronic inflammatory diseases? \u003cem\u003eEur. J. Immunol.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1098\u0026ndash;1108 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLimmathurotsakul, D. et al. Increasing Incidence of Human Melioidosis in Northeast Thailand. \u003cem\u003eAm. Soc. Trop. Med. Hygiene\u003c/em\u003e. \u003cb\u003e82\u003c/b\u003e, 1113\u0026ndash;1117 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaukonen, K. M., Bailey, M., Pilcher, D., Cooper, D. J. \u0026amp; Bellomo, R. Systemic inflammatory response syndrome criteria in defining severe sepsis. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e372\u003c/b\u003e, 1629\u0026ndash;1638 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"mucosal associated invariant T cells, sepsis, melioidosis, diabetes","lastPublishedDoi":"10.21203/rs.3.rs-9083519/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9083519/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBurkholderia pseudomallei\u003c/em\u003e (BP), the causative agent of melioidosis, is a major cause of sepsis in Southeast Asia, especially in people with diabetes mellitus (DM). The role of Mucosal-associated invariant T (MAIT) cells; innate-like T cells important for antibacterial immunity; in melioidosis is unknown.\u003c/p\u003e \u003cp\u003eWe measured MAIT cell activation by BP \u003cem\u003ein vitro\u003c/em\u003e using co-culture assays with THP-1 cells, and evaluated MAIT cell frequency, activation, and function \u003cem\u003eex vivo\u003c/em\u003e in an observational cohort (n\u0026thinsp;=\u0026thinsp;120) of melioidosis patients and endemic controls with and without DM in Thailand.\u003c/p\u003e \u003cp\u003eWe show that BP induces IFN-γ secretion by MAIT cells in a cytokine dependent manner. In acute melioidosis, circulating MAIT cells, particularly the double-negative (DN) subset, were significantly reduced, and highly activated but dysfunctional, with reduced IFN-γ responses to BP and \u003cem\u003eE. coli\u003c/em\u003e which were restored upon recovery. Among acute patients, non-survivors showed lower granzyme B and IFN-γ expression. Acute melioidosis patients with DM co-morbidity exhibited reduced DN MAIT cell frequency and responses to \u003cem\u003eE. coli\u003c/em\u003e compared to non-DM patients.\u003c/p\u003e \u003cp\u003eOverall, the frequency and function of MAIT cells is impaired during acute melioidosis, especially in patients with DM, indicating a key role for these cells in antibacterial defence and disease susceptibility.\u003c/p\u003e","manuscriptTitle":"Mucosal-associated invariant T (MAIT) cells are reduced and dysfunctional in acute melioidosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 06:46:23","doi":"10.21203/rs.3.rs-9083519/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-08T06:38:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T04:26:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74184432581156099105914947101612554549","date":"2026-05-06T06:48:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T09:26:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182556496040165263144093023103340460791","date":"2026-04-10T08:21:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T01:12:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T01:09:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-18T14:58:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T13:30:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-16T10:50:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f321a9c7-f9dc-4fcf-b1a2-ab0884c404ee","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-08T06:38:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T04:26:09+00:00","index":89,"fulltext":""},{"type":"reviewerAgreed","content":"74184432581156099105914947101612554549","date":"2026-05-06T06:48:21+00:00","index":88,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66169523,"name":"Health sciences/Diseases"},{"id":66169524,"name":"Biological sciences/Immunology"},{"id":66169525,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-08T06:55:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 06:46:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9083519","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9083519","identity":"rs-9083519","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}