A case–control study was conducted to estimate the prevalence of leptospirosis in Tiruchirappalli, to determine the serum levels of MIF in leptospirosis patients compared with other febrile cases and healthy control subjects, and to assess the leptospiral lipopolysaccharide (LPS)-mediated MIF upregulation in in vitro and in vivo experimental models. This study was carried out from June 2017 to February 2018 by active hospital-based surveillance at the Annal Gandhi Memorial General Hospital, Tiruchirappalli, Tamil Nadu, India. The temperature in the study area ranges from 36°C to 41°C, and its geographical position is 10°48′18″ N latitude and 78°41′08″ E longitude.

A total of 300 study subjects were recruited to participate in the study. In total, 243 blood samples from clinically suspected cases with clinical manifestations such as fever, myalgia, body ache, arthritis, icterus, rigors, breathlessness, abdominal pain, conjunctival suffusion, subconjunctival hemorrhages, and jaundice with acute renal failure were collected for the diagnosis of leptospirosis. The samples were collected before any treatment was given to the patients. A total of 57 seronegative healthy controls recruited from the general population in the same geographical area matched for age ( ± 5 years) and sex were included as controls. Healthy control subjects who had fever in the previous 2 weeks were excluded from the study. The obtained serum samples were divided into aliquots and stored at −80°C until the assay was performed.

For serological evidence of leptospirosis in the study population, microscopic agglutination test (MAT) was performed using a panel of 12 live leptospiral serovars. Leptospiral cultures were maintained by regular sub-culturing in Ellinghausen–McCullough–Johnson–Harris (EMJH) medium supplemented with bovine serum albumin and Tween-80 at the Medical Microbiology Laboratory, Bharathidasan University, Tiruchirappalli. The following serogroups were used as live antigens: Australis (serovar Australis, strain Ballico), Autumnalis (serovar Autumnalis, strain Akiyami A), Ballum (serovar Ballum, strain Mus 127), Bataviae (serovar Bataviae, strain Swart), Canicola (serovar Canicola, strain Hond Utrecht IV), Icterohaemorrhagiae (serovar Icterohaemorrhagiae, strain RGA), Grippotyphosa (serovar Grippotyphosa, strain Moskva V), Hebdomadis (serovar Hebdomadis, strain Hebdomadis), Javanica (serovar Poi, strain Poi), Pomona (serovar Pomona, strain Pomona), Pyrogenes (serovar Pyrogenes, strain Salinem), and Sejroe (serovar Hardjo, strain Hardjoprajitno). Seven-day-old live leptospiral culture of 1 × 10⁸ organisms/ml was used as the antigen. Double dilution of serum was performed serially starting from 1:20 and incubated with live antigens for agglutination. A titer of ≥1:160 and agglutination of ≥50% were considered as positive for MAT. Phosphate-buffered saline (PBS) was used as a diluent in the assay.

Immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was performed to further confirm leptospiral infection in suspected cases by assessing the early immune response of cases to leptospirosis. Heat-extracted leptospiral antigens were prepared as described earlier (Kanagavel et al., 2017). Of the leptospiral antigens, 0.2 µg was coated on 96-well microtiter plates at appropriate wells using carbonate coating buffer (pH 9.6) and then stored at 4°C for 12 h. Each well was washed three times with PBST (PBS + 0.1% Tween-20) for 10 min each. About 3% blocking solution (non-fat milk) was added to each well and incubated at 37°C for 1 h. Each well was washed, as previously mentioned. The test sera were added into appropriate wells at a dilution of 1:100 and incubated at 37°C for 1 h. After washing the wells, the bound IgM antibody was detected by adding peroxide-conjugated anti-human IgM antibody (1:1,000) and incubated at 37°C for 1 h, followed by developing with o-phenylenediamine dihydrochloride (OPD). Fifty microliters of 1 N H₂SO₄ was added to stop the reaction, and then the optical density was measured at 490 nm using a microtiter plate reader.

Quantitative measurement of human MIF in patient sera was carried out using Human MIF ELISA Kit (Sigma-Aldrich, St. Louis, Mo, USA). All procedures were in accordance with the manufacturer’s instructions. In brief, all reagents and samples were allowed to reach 18–25°C before use. All samples and standards were performed in triplicate. Of the samples, 100 μl was added into microtiter wells and incubated for 3 h at room temperature with gentle shaking. Then, the solution was discarded and the wells washed four times with 1× wash buffer. Subsequently, 100 μl of 1× biotinylated detection antibody was added to each well and incubated for 1 h at room temperature with gentle shaking. Following washing, 100 μl of horseradish peroxidase (HRP)–streptavidin solution was added to each well and incubated for 45 min at room temperature with gentle shaking. The wells were again washed and 100 μl of ELISA colorimetric 3,3′,5,5′-tetramethylbenzidine (TMB) reagent was added to each well and incubated for 30 min at room temperature in the dark. Fifty microliters of the stop reagent was added to each well and the plates immediately read at 450 nm. The mean absorbance values for each set of standards, controls, and samples were calculated and the average zero standard optical densities were subtracted for background correction. The standard curve was plotted using SigmaPlot 11.0 software, with the standard concentration on the x-axis and absorbance on the y-axis to quantify the MIF. Serum MIF levels were expressed as nanograms per milliliter.

Mouse-adapted challenge strains (MACS) were prepared from their corresponding parent strains (reference laboratory strains/isolates) by passaging them in immunocompromised BALB/c mice (300 mg/kg body weight of cyclophosphamide treatment) ~15 times (Adler and Faine, 1976; Adler and Faine, 1977). The cultures used in all the experiment in the current study were Leptospira interrogans serovar Autumnalis strain N2 (human isolate) MACS passaged in vitro less than three times (Kanagavel et al., 2017).

L. interrogans serovar Autumnalis strain N2 MACS were grown in liquid EMJH medium at 30°C and collected at a density of ~5 × 10⁸ leptospires/ml. Leptospiral LPS was extracted with the standard hot phenol–water method (Westphal and Jann, 1965). The phenol phase was purified by dialysis. The extracted LPS were quantified using the phenol/sulfuric acid method (Vanithamani et al., 2015).

THP-1 cells were purchased from the National Centre for Cell Science, Pune, and cultured in RPMI 1640 medium with 10% fetal bovine serum at 37°C and 5% CO₂. The cells were pelleted and washed three times with serum-free culture medium. Approximately 5 × 10⁵ cells per well were added into six-well plates and then treated with LPS (1 μg/ml) at different time intervals (0, 5, 10, 20, and 30min and 1, 2, 3, 6, 12, 24, 48, and 60 h). All treatments were performed in triplicate. Treated cells were pelleted for further analysis.

An inbred strain of BALB/c mice (weighing about 20 ± 2 g) was used throughout the study. To accustom the mice, they were housed under ambient room temperature (25 ± 2°C), with a 12-h light/dark cycle, and given standard feed and water ad libitum over a period of 10 days before the start of the experiments. Mice 4–6 weeks old were separated into five groups. Five mice (immunocompromised) in each group were challenged (intraperitoneally) with different doses (5, 10, 20, and 30 mg/kg) of L. interrogans serovar Autumnalis strain N2 for the determination of the survivability of the BALB/c mouse model. Mice injected with PBS were considered as untreated control. Survival over 4 days was frequently evaluated and the LD₅₀ of leptospiral LPS determined.

Dot blot assay and cytokine profiling were performed to examine endotoxemia in LD₅₀ LPS-injected mice. Blood sample was collected from the control (PBS-injected) and LPS-injected mice and the plasma separated for analysis. Two miroliters of plasma samples and 5 µg purified LPS were loaded into a 0.2-µm nitrocellulose membrane, probed with patient sera (1:100), and incubated for 1 h at room temperature. Anti-human IgM (ALP-conjugated) was added to the membrane and incubated for 1 h. The membrane was washed for 30 min with PBST after every incubation step, developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), and documented in Fusion Solo™ Personal Blot and Gel Imaging System (Vilber Lourmat, Paris, France). Dot intensity was measured densitometrically and expressed in arbitrary units.

Quantitative real-time PCR (qRT-PCR) was carried out to measure the expressions of the inflammatory cytokines [tumor necrosis factor alpha (TNF-α), IL-1β, IL-4, and IL-10] of mice injected with LD₅₀ LPS. Monolayer cells from the plasma were collected and RNA was isolated with the RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). Complementary DNA (cDNA) was synthesized and qRT-PCR was performed using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The primers used in this study are listed in Supplementary Table S1 .

The mice to be injected with leptospiral LPS were primarily immunocompromised with cyclophosphamide (300 mg/kg body weight). The mice were subjected to LPS exposure by intraperitoneal injection. The untreated control group was maintained by injecting with PBS alone. The dosage of LPS was 20 mg/kg body weight. Infected mice were monitored every 12 h for clinical outcomes and survival up to 8 days. Dead and moribund animals were euthanized for harvesting the tissues. Vital organs including the heart, lungs, kidney, spleen, and liver and lymphoid organs such as the thymus, bone marrow, spleen, and lymph node were collected and stored in liquid nitrogen for MIF profiling experiments.

The collected THP-1 cells and homogenized tissues were washed three times with ice-cold PBS. Cell lysates were prepared using ice-cold RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1 mM EDTA, and 1% NP-40) (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20°C. The extracted protein was quantified using the bicinchoninic acid (BCA) method. Thirty micrograms of protein per well was loaded onto 12% polyacrylamide gel and electrophoresed at 60 V in Mini-PROTEAN Tetra System (Bio-Rad, Hercules, CA, USA). The separated proteins were transferred into a nitrocellulose membrane (pore size, 0.2 µm) electrophoretically at 12 V for 1 h using a V20 semi-dry blotter (Scie-Plas, Cambridge, UK). Subsequently, the membranes were blocked with a blotting grade blocking solution (5%, w/v) at room temperature for 1 h, washed three times for 10 min each with 1× TBST (TBS + 0.1% Tween-20), and incubated with rabbit anti-MIF antibody (1:1,000; Invitrogen, Carlsbad, CA, USA) at 4°C for 12 h. Bound antibodies were detected by incubating with HRP-conjugated anti-rabbit antibody (1:5,000; Sigma-Aldrich, St. Louis, MO, USA). Bands were developed with the West Pico Signal Chemiluminescence developing kit (Thermo Fisher Scientific, Waltham, MA, USA) and documented in Fusion Solo™ Personal Blot and Gel Imaging System (Vilber Lourmat, Paris, France). Band intensities were calculated using ImageJ software, and the data were normalized to the loading control.

RNA was extracted from the harvested tissues and THP-1 cells using an RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). The concentration and the purity of mRNA were determined using the BioPhotometer Plus system (Eppendorf, Hamburg, Germany). cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) The primers used in this study are listed in Supplementary Table S1 . The qRT-PCR using SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) and primers was carried out in a 10-μl reaction volume (20 ng cDNA, 5 μl Master Mix, and 0.2 μM of each primer). GAPDH was used as the loading control.

Data from triplicate experiments were quantified and expressed as the mean ± SD (n = 3). Serum MIF levels with outlier points were expressed as median (interquartile range, IQR). Data were computed either with GraphPad Prism version 9.2.0 or SigmaPlot 11.0 software. Receiver operating characteristic (ROC) analyses were performed to estimate the sensitivity and specificity of MIF as a diagnostic marker for leptospirosis. Pearson’s correlation coefficient test was performed to assess the correlation of serum MIF with disease duration and patient age. A two-tailed paired Student’s t-test or the Mann–Whitney U test was performed to analyze differences between the study groups. Kaplan–Meier plots were generated using GraphPad Prism version 7.0 to quantify survivability. A p-value ≤0.05 was considered significant.

The studies involving human participants (for the collection of blood samples) were reviewed and approved by the Institutional Ethical Committee (no. DM/2014/101/51) of Bharathidasan University. Informed consent was obtained from both patients and healthy controls prior to the sample collection; in the case of minor study participants, their surrogates signed an informed consent form. The animal experimental protocols were approved by the Institutional Animal Ethical Committee (BDU/IAEC/P30/2018), Bharathidasan University.

The seroprevalence of leptospirosis was determined with the MAT assay with the panel of 12 leptospiral serovars as an antigen. Out of the 300 serum samples tested for antibodies against pathogenic Leptospira, a total of 142 cases tested positive at agglutination titer of ≥1:80, giving an overall seroprevalence of 47.3%. Seropositivity was uniformly distributed in both genders and in all age groups. The demographic characteristics of the clinical subjects included in the study are shown in Table 1 .

Of the 12 leptospiral serovars tested, six were detected among the samples from the study subjects. The prevalent infecting serovars were Autumnalis (50.7%), followed by Australis (21.2%), Canicola (16.2%), Icterohaemorrhagiae (15.6%), Grippotyphosa (3.5%), and Ballum (2.9%). MAT titers were reported to be in the range between 1:80 and 1:2,560. The MAT-positive titers with the respective serovars are represented in Table 2 . Among the 142 patients who tested positive for MAT, 9 (6.3%) cases were negative for IgM ELISA.

To identify whether MIF protein was differentially expressed between leptospirosis patients and healthy control subjects, we used the sera of the study subjects to assess circulating MIF concentrations with MIF ELISA. The cutoff point for a positive MIF level was 0.008 ng/ml. As shown in Figure 1A , it was found that the serum MIF levels were significantly (p < 0.001) elevated in different clinical conditions of patients with leptospirosis, including febrile illness (median = 7.5 ng/ml, IQR = 5.32–8.97), pulmonary hemorrhage (median = 13.2 ng/ml, IQR = 11.77–16.72), Weil’s syndrome (median = 8.8 ng/ml, IQR = 7.25–9.95), and renal failure (median = 8.65 ng/ml, IQR = 7.18–10.5), compared to those in healthy controls (median = 0.65 ng/ml, IQR = 0.5–1.1) and other febrile cases such as typhoid (median = 1.32 ng/ml, IQR = 0.57–1.9), malaria (median = 1.1 ng/ml, IQR = 0.57–1.35), dengue (median = 2.2 ng/ml, IQR = 1.58–2.8), hepatitis (median = 1.32 ng/ml, IQR = 0.62–1.56), syphilis (median = 1.9 ng/ml, IQR = 1.46–2.2), shigellosis (median = 1.31 ng/ml, IQR = 0.67–1.5), and enteritis (median = 1.2 ng/ml, IQR = 0.8–1.85). Patients with pulmonary hemorrhage have remarkably higher MIF levels than with other clinical manifestations. The MIF levels of all study subjects are presented in Table 3 .

To estimate the diagnostic value of serum MIF as a biomarker for leptospirosis, ROC curve analysis was performed. ROC analysis revealed that the serum MIF levels discriminated significantly between leptospirosis cases and healthy control subjects. The area under the curve (AUC) of the different clinical manifestations of leptospirosis was >0.9 (p < 0.0001) ( Supplementary Figures S1A–D ). An AUC value >0.9 was considered as an outstanding quality of discrimination between groups. The optimal cutoff value for MIF was predicted as 2.5 ng/ml by stressing the higher sensitivity. The sensitivity and the specificity of serum MIF profiling for leptospirosis cases were found to be 100% and >90%, respectively, in different clinical forms of leptospirosis. The positive predictive value (PPV) and the negative predictive value (NPV) were >90% and 100%, respectively. The sensitivity/specificity values and the PPV/NPV for the different clinical manifestations of leptospirosis are shown in Table 4 . Our results demonstrated that the levels of serum MIF were significantly upregulated in leptospirosis patients compared to those in healthy control subjects and that it will be possible to use MIF as a biomarker to improve disease monitoring and management.

The difference in the serum MIF levels between leptospirosis cases with febrile illness (mild, non-hospitalized) and those with organ dysfunction (severe, hospitalized) was significantly high (2.5, p < 0.001). Our results revealed that the serum MIF levels were more significantly elevated in patients with severe than in those with mild leptospirosis. Here, we analyzed whether there was any association between serum MIF and leptospirosis disease progression. We evaluated the serum MIF levels associated with disease progression using Pearson’s correlation test and found a significantly high positive correlation (r = 0.75, p < 0.0001). A correlation coefficient index (r) of ≥0.7 was considered as a high positive correlation. There was no significant correlation found in the serum MIF levels of study subjects in respect to age (r = 0.006). An r value of <0.3 was considered as negligible correlation between variables. The correlation of elevated serum MIF with the disease progression of leptospirosis is represented in Figure 1B . The analysis suggested that serum MIF may serve as a severity predictor of human leptospirosis.

For the determination of LD₅₀, LPS-challenged mice were frequently evaluated for disease progression and moribund state. The number of deceased mice was recorded to plot the Kaplan–Meier survival graph. The LD₅₀ for leptospiral LPS was found to be 20 mg/kg, which caused the death of about 50% of the mice. Mice challenged with 5, 10, and 30 mg/kg of LPS exhibited 80%, 80%, and 20% of survival, respectively ( Figure 2A ). Thus, we used 20 mg/kg of leptospiral LPS for subsequent experiments.

The dot blot immunoassay detected the significant occurrence of LPS in the blood stream of infected mice. This confirmed that the intraperitoneal injection of LD₅₀ LPS induced endotoxemia in the mouse model ( Figures 2B, C ). Endotoxemic inflammation after injection of LD₅₀ LPS was characterized by analysis of the cytokine expressions. The qRT-PCR results showed a significant (15-fold) increase of the expressions of TNF-α and IL-1β and a threefold increase of the expression of IL-4 ( Figure 2D ). The high expressions of pro-inflammatory cytokines in LPS-infected mice revealed endotoxemic inflammation. Therefore, the administration of leptospiral LD₅₀ LPS induced endotoxemic inflammation in BALB/c mice.

To demonstrate the MIF expression profile during the early phase of leptospiral infection, we have experimentally induced leptospiral infection in in vitro and in vivo models. Cell lysates prepared from THP-1 cells treated with leptospiral LPS (1 μg/ml) were assayed using Western blotting to determine the intracellular MIF profile ( Figures 3A, D ). MIF expression was at the baseline in untreated cells. Leptospiral LPS significantly (p < 0.001) increased the expression level of MIF in a time-dependent manner. The expression of MIF in THP-1 cells was markedly increased at 30 min post-treatment, which gradually upregulated and reached the peak level at 24 h post-treatment and then significantly diminished at 60 h.

To assess the elevated MIF expression in lymphoid organs and other vital organs during leptospiral pathology, tissue samples were harvested from leptospiral LPS-administered BALB/c mice and were assayed with Western blotting. The results showed significantly (p < 0.01) upregulated MIF expression in vital organs post-LPS injection, particularly in the lung, spleen, and heart compared to other organs ( Figures 3B, E ). As MIF is an immune mediator, we investigated the regulation of its expression in both primary and secondary lymphoid organs, including the thymus, bone marrow, spleen, and lymph node, on days 0, 1, 2, 3, and 4 post-LPS treatment. The expression of MIF was significantly increased in all lymphoid organs on days 1, 2, 3, and 4 post-LPS treatment when compared with day 0. In untreated control mice, differential MIF expression was observed in vital organs, whereas no significant difference in its expression was noticed between lymphoid organs ( Figures 3C, F ).

Furthermore, RT-PCR analysis confirmed the LPS-mediated upregulated MIF gene expression by measuring the increased MIF mRNA levels. Leptospiral LPS gradually upregulated the MIF gene expression in a time-dependent manner in LPS-treated THP-1 cells ( Figure 3G ). As in the vitro MIF protein profile, the MIF gene expression was remarkably increased in the lung, spleen, and heart of LPS-injected mice ( Figure 3H ). MIF gene expression analysis on lymphoid organs at different time intervals revealed that the mRNA expression was gradually increased in all lymphoid organs up to day 3 and drastically increased on day 4. No significant morphological changes were observed in the lymphoid organs of mice injected with LD₅₀ LPS ( Figure 3I ). These results confirmed that MIF is an early-phase secreted cytokine and that its use as a biomarker will be a promising early diagnostic tool for leptospirosis. Taken together, the mRNA and protein expressions of MIF were significantly increased as the duration of infection increased, with MIF possibly driving the pathogenesis of leptospirosis.