• View in gallery
    Figure 1.

    Neutrophil activation was not associated with disease severity. Expression of CD62L (A) and f CD11b (B) on neutrophils during acute leptospirosis indicate activation levels similar to those in convalescent patients and healthy controls. Results are shown as the percent of granulocytes positive for CD15 and CD62L (A) or CD11b (B) and MFI of CD62L (A) or CD11b (B) on CD15+ granulocytic cells. Groups are A (acute leptospirosis, no renal/lung dysfunction; n = 6), S (severe acute leptospirosis, renal/lung dysfunction; n = 4), C (convalescent time point; n = 4), and H (healthy controls; n = 4). ns indicates no significant differences in group comparisons (nonparametric one-way ANOVA). Means ± SEM are shown.

  • View in gallery
    Figure 2.

    Neutrophil activation function was not associated with disease group. Cells were stimulated with toll-like receptor agonists as described. Results are represented as the means ± SEM for MFI fold-changes of CD62L (A) and CD11b (B) on CD15+ granulocytes. A value of “1” indicates no response to agonist stimulation relative to unstimulated controls. Groups are A (acute leptospirosis, no renal/lung dysfunction; n = 6), S (severe acute leptospirosis, renal/lung dysfunction; n = 4), and C (convalescent time point; n = 4). ns indicates no significant differences in group comparisons (nonparametric one-way ANOVA). Means ± SEM are shown.

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    Figure 3.

    Increased toll-like receptor (TLR) expression on neutrophils in acute leptospirosis patients with renal/lung dysfunction. (A) Increased TLR2 expression in patients with renal and/or lung dysfunction (group S). (B) TLR4 expression on neutrophils was lower than TLR2 expression but displayed a similar expression pattern across groups. Results are represented as the percent granulocytes positive for CD15 and TLR2 (A) or TLR4 (B) after isotype subtraction. Groups are A (acute leptospirosis, no renal/lung dysfunction; n = 10), S (severe acute leptospirosis, renal/lung dysfunction; n = 5), C (convalescent time point; n = 5), and H (healthy Brazilian adults; n = 4). Asterisks indicate significant differences relative to group S (*P < 0.05, **P < 0.01). Means ± SEM are shown. No other significant differences between groups were identified.

  • 1.

    Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-Ridder B, Ko AI, 2015. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 9: e0003898.

    • Search Google Scholar
    • Export Citation
  • 2.

    Ko AI, Goarant C, Picardeau M, 2009. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7: 736747.

    • Search Google Scholar
    • Export Citation
  • 3.

    Gouveia EL, Metcalfe J, de Carvalho AL, Aires TS, Villasboas-Bisneto JC, Queirroz A, Santos AC, Salgado K, Reis MG, Ko AI, 2008. Leptospirosis-associated severe pulmonary hemorrhagic syndrome, Salvador, Brazil. Emerg Infect Dis 14: 505508.

    • Search Google Scholar
    • Export Citation
  • 4.

    Vieira SR, Brauner JS, 2002. Leptospirosis as a cause of acute respiratory failure: clinical features and outcome in 35 critical care patients. Braz J Infect Dis 6: 135139.

    • Search Google Scholar
    • Export Citation
  • 5.

    Marotto PC, Nascimento CM, Eluf-Neto J, Marotto MS, Andrade L, Sztajnbok J, Seguro AC, 1999. Acute lung injury in leptospirosis: clinical and laboratory features, outcome, and factors associated with mortality. Clin Infect Dis 29: 15611563.

    • Search Google Scholar
    • Export Citation
  • 6.

    Murgia R, Garcia R, Cinco M, 2002. Leptospires are killed in vitro by both oxygen-dependent and independent reactions. Infect Immun 70: 71727175.

    • Search Google Scholar
    • Export Citation
  • 7.

    Linde A, Lushington GH, Abello J, Melgarejo T, 2013. Clinical relevance of cathelicidin in infectious disease. J Clin Cell Immunol S13: 003.

  • 8.

    Scharrig E, Carestia A, Ferrer MF, Cedola M, Pretre G, Drut R, Picardeau M, Schattner M, Gomez RM, 2015. Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PLoS Negl Trop Dis 9: e0003927.

    • Search Google Scholar
    • Export Citation
  • 9.

    Pillai PS et al. 2016. Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease. Science 352: 463466.

  • 10.

    De Silva NL, Niloofa M, Fernando N, Karunanayake L, Rodrigo C, De Silva HJ, Premawansa S, Handunnetti SM, Rajapakse S, 2014. Changes in full blood count parameters in leptospirosis: a prospective study. Int Arch Med 7: 31.

    • Search Google Scholar
    • Export Citation
  • 11.

    Craig SB, Collet TA, Wynwood SJ, Smythe LD, Weier SL, McKay DB, 2013. Neutrophil counts in leptospirosis patients infected with different serovars. Trop Biomed 30: 579583.

    • Search Google Scholar
    • Export Citation
  • 12.

    Craig SB, Graham GC, Burns MA, Dohnt MF, Smythe LD, McKay DB, 2009. Haematological and clinical-chemistry markers in patients presenting with leptospirosis: a comparison of the findings from uncomplicated cases with those seen in the severe disease. Ann Trop Med Parasitol 103: 333341.

    • Search Google Scholar
    • Export Citation
  • 13.

    Raffray L, Giry C, Vandroux D, Kuli B, Randrianjohany A, Pequin AM, Renou F, Jaffar-Bandjee MC, Gasque P, 2016. Major neutrophilia observed in acute phase of human leptospirosis is not associated with increased expression of granulocyte cell activation markers. PLoS One 11: e0165716.

    • Search Google Scholar
    • Export Citation
  • 14.

    Futosi K, Fodor S, Mocsai A, 2013. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol 17: 638650.

    • Search Google Scholar
    • Export Citation
  • 15.

    Zuerner RL, 2015. Host response to Leptospira infection. Curr Top Microbiol Immunol 387: 223250.

  • 16.

    Nahori MA, Fournie-Amazouz E, Que-Gewirth NS, Balloy V, Chignard M, Raetz CR, Saint Girons I, Werts C, 2005. Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells. J Immunol 175: 60226031.

    • Search Google Scholar
    • Export Citation
  • 17.

    Werts C et al. 2001. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat Immunol 2: 346352.

  • 18.

    Goris MG, Wagenaar JF, Hartskeerl RA, van Gorp EC, Schuller S, Monahan AM, Nally JE, van der Poll T, van’t Veer C, 2011. Potent innate immune response to pathogenic Leptospira in human whole blood. PLoS One 6: e18279.

    • Search Google Scholar
    • Export Citation
  • 19.

    Viriyakosol S, Matthias MA, Swancutt MA, Kirkland TN, Vinetz JM, 2006. Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden. Infect Immun 74: 887895.

    • Search Google Scholar
    • Export Citation
  • 20.

    Chassin C et al. 2009. TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans. J Immunol 183: 26692677.

    • Search Google Scholar
    • Export Citation
  • 21.

    Guo Y, Fukuda T, Donai K, Kuroda K, Masuda M, Nakamura S, Yoneyama H, Isogai E, 2015. Leptospiral lipopolysaccharide stimulates the expression of toll-like receptor 2 and cytokines in pig fibroblasts. Anim Sci J 86: 238244.

    • Search Google Scholar
    • Export Citation
  • 22.

    Guo Y, Fukuda T, Nakamura S, Bai L, Xu J, Kuroda K, Tomioka R, Yoneyama H, Isogai E, 2015. Interaction between leptospiral lipopolysaccharide and toll-like receptor 2 in pig fibroblast cell line, and inhibitory effect of antibody against leptospiral lipopolysaccharide on interaction. Asian-Australas J Anim Sci 28: 273279.

    • Search Google Scholar
    • Export Citation
  • 23.

    Zhang W, Zhang N, Xie X, Guo J, Jin X, Xue F, Ding Z, Cao Y, 2016. Toll-like receptor 2 agonist Pam3CSK4 alleviates the pathology of leptospirosis in hamster. Infect Immun 84: 33503357.

    • Search Google Scholar
    • Export Citation
  • 24.

    Chen X, Li SJ, Ojcius DM, Sun AH, Hu WL, Lin X, Yan J, 2017. Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis. PLoS One 12: e0181014.

    • Search Google Scholar
    • Export Citation
  • 25.

    Stoddard RA, Gee JE, Wilkins PP, McCaustland K, Hoffmaster AR, 2009. Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene. Diagn Microbiol Infect Dis 64: 247255.

    • Search Google Scholar
    • Export Citation
  • 26.

    Smythe LD, Smith IL, Smith GA, Dohnt MF, Symonds ML, Barnett LJ, McKay DB, 2002. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infect Dis 2: 13.

    • Search Google Scholar
    • Export Citation
  • 27.

    Qian F, Guo X, Wang X, Yuan X, Chen S, Malawista SE, Bockenstedt LK, Allore HG, Montgomery RR, 2014. Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging. Aging (Albany NY) 6: 131139.

    • Search Google Scholar
    • Export Citation
  • 28.

    Wang B, Sullivan J, Sullivan GW, Mandell GL, 1984. Interaction of leptospires with human polymorphonuclear neutrophils. Infect Immun 44: 459464.

    • Search Google Scholar
    • Export Citation
  • 29.

    Lindow JC et al. 2016. Cathelicidin insufficiency in patients with fatal leptospirosis. PLoS Pathog 12: e1005943.

  • 30.

    Arean VM, 1962. The pathologic anatomy and pathogenesis of fatal human leptospirosis (Weil’s disease). Am J Pathol 40: 393423.

  • 31.

    Sitprija V, Evans H, 1970. The kidney in human leptospirosis. Am J Med 49: 780788.

  • 32.

    Jaillon S, Galdiero MR, Del Prete D, Cassatella MA, Garlanda C, Mantovani A, 2013. Neutrophils in innate and adaptive immunity. Semin Immunopathol 35: 377394.

    • Search Google Scholar
    • Export Citation
  • 33.

    Reis EA, Hagan JE, Ribeiro GS, Teixeira-Carvalho A, Martins-Filho OA, Montgomery RR, Shaw AC, Ko AI, Reis MG, 2013. Cytokine response signatures in disease progression and development of severe clinical outcomes for leptospirosis. PLoS Negl Trop Dis 7: e2457.

    • Search Google Scholar
    • Export Citation
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Elevated Activation of Neutrophil Toll-Like Receptors in Patients with Acute Severe Leptospirosis: An Observational Study

Janet C. LindowDepartment of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut;
Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;

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Annie J. TsayDepartment of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut;

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Ruth R. MontgomerySection of Rheumatology, Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut;

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Eliana A. G. ReisGonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;

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Elsio A. Wunder Jr.Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut;
Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;

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Guilherme AraújoGonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;

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Nivison R. R. Nery Jr.Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;

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Subhasis MohantySection of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut;

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Albert C. ShawSection of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut;

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Patty J. LeeSection of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut;

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Mitermayer G. ReisDepartment of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut;
Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;
Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Salvador, Brazil

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Albert I. KoDepartment of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut;
Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil;

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Leptospirosis is the leading cause of zoonotic morbidity and mortality globally, yet little is known about the immune mechanisms that may contribute to pathogenesis and severe disease. Although neutrophils are a key component of early immune responses to infection, they have been associated with tissue damage and inflammation in some febrile infections. To assess whether neutrophils contribute to the pathogenesis observed in severe leptospirosis, we quantitated levels of neutrophil activation markers in patients with varying disease severities. Hospitalized leptospirosis patients had significantly higher levels of toll-like receptors 2 and 4 (TLR2 and TLR4, respectively) on peripheral neutrophils than healthy controls, with the highest levels detected in patients with organ dysfunction. We observed no significant differences in other neutrophil baseline activation markers (CD62L and CD11b) or activation capacity (CD62L and CD11b levels following stimulation), regardless of disease severity. Our results provide preliminary evidence supporting the hypothesis that higher initial bacterial loads or inadequate or delayed neutrophil responses, rather than TLR-driven inflammation, may drive severe disease outcomes.

Leptospirosis is a neglected zoonotic disease of global importance with more than one million cases and nearly 60,000 deaths occurring annually, primarily in slum settlements, worldwide.1 In Salvador, Brazil, large epidemics of leptospirosis, usually caused by Leptospira interrogans serovar Copenhageni, occur during seasonal periods of heavy rainfall.2,3 The bacteria penetrate skin or mucous membranes, with clinical manifestations ranging from mild to severe flu-like symptoms to multi-organ failure and death.2 Notably, Weil’s disease, the classic severe form of leptospirosis, characterized by jaundice, acute renal failure and bleeding, and Leptospira-associated pulmonary hemorrhage syndrome have case fatalities of > 10% and 50%, respectively.35

Neutrophils are abundant innate immune cells capable of killing extracellular Leptospira via phagocytosis, production of reactive oxygen and nitrogen species, neutrophil extracellular traps, and specific antimicrobial peptides.68 Neutrophils at the site of infection help initiate adaptive immune responses through production of cytokines and chemokines. Despite their critical role in resolving infections, neutrophils have been associated with exacerbation of lung infections in influenza and other nosocomial infections,9 and could contribute to the inflammation leading to organ damage observed in 10–15% of leptospirosis cases, particularly as higher neutrophil counts have been observed in patients with more severe disease.2,1012 However, a recent study of neutrophils in hospitalized cases showed that neutrophils were not activated during acute leptospirosis despite patients having significant neutrophilia.13

Neutrophils recognize conserved microbial motifs through pattern recognition receptors, such as toll-like receptors (TLRs), which trigger activation of antimicrobial responses and production of pro-inflammatory cytokines.14 To date, in vitro experiments using transfected human cell lines and healthy volunteer peripheral blood mononuclear cells (PBMCs) or whole blood have yielded contradictory results regarding the involvement of TLR2 and TLR4 in the human immune response to the leptospiral lipopolysaccharide (LPS).1518 A single study in patients showed neutrophils expressed TLR2 during acute leptospirosis, but did not analyze TLR4 expression.13 Results from animal models of leptospirosis have yielded differing TLR responses to leptospiral LPS, with control of infection mediated by TLR2 and TLR4 in mice16,17,19,20 and activation of TLR2 in pig cell lines.21,22 A separate study demonstrated that resistant mice, but not hamsters, induced TLR2 early in infection, and when Leptospira and a TLR2 agonist were co-injected in hamsters, pathology and survival improved.23 In addition, a recent study showed mononuclear macrophages had greater capacity for killing Leptospires than neutrophils and were present in larger abundance in organ infiltrates in mice with leptospirosis.24 We have undertaken the present study to examine TLR expression and function on neutrophils during acute human leptospirosis to identify whether neutrophil activation is associated with organ dysfunction and severe outcomes.

Between July 2013 and August 2014, we identified patients with suspected leptospirosis through active surveillance at a state-run infectious disease hospital in Salvador, Brazil. We confirmed 15 (79%) of 19 serially recruited cases, using at least one of the criteria described: seroconversion or 4-fold rise in titer in paired sera samples or titer ≥ 1:800 in a single sera sample measured by serum micro-agglutination test, positive blood quantitative polymerase chain reaction (qPCR) (Leptospira load/mL blood), or positive blood culture.3,25,26 We gathered clinical data during patient interviews and from hospital charts and collected paired venous blood samples from patients during acute illness (n = 18 within 72 hours of hospital admission and n = 1 within 96 hours) and paired convalescence samples from four individuals not lost to follow-up (32–57 days post-admission). In addition, we obtained samples from four healthy Brazilian adults. We categorized the 15 confirmed patients into two groups based on organ dysfunction: severe group “S” patients with evidence of either renal dysfunction (oliguria (< 500 mL urine/day) or anuria (< 50 mL urine/day) within 24 hours of hospital admission) and/or lung injury during hospitalization (mechanical ventilation and/or requiring oxygen, ≥ 250 mL blood in lungs or endotracheal tube, and/or respiration rate > 38/minutes), whereas acute group “A” patients met none of these criteria (Table 1).

Table 1

Laboratory and clinical findings at admission for patients with confirmed leptospirosis (n = 15)

DemographicsNo organ dysfunction (Acute [A])*Organ dysfunction (severe [S])†
No.Median (IQR) or no. (%)No.Median (IQR) or no. (%)P-value‡
Gender (male)108 (80%)55 (100%)0.524
Age (years)1030.0 (18.8–39.8)531.0 (23.5–39.5)0.799
Clinical presentation
 Days of illness§106.5 (5.0–7.3)56.0 (3.5–6.5)0.170
 Jaundice107 (70%)55 (100%)0.506
Clinical laboratory‖
 Hematocrit (%)1033.6 (31.5–40.6)535.4 (34.7–37.8)0.699
 Leukocyte count (1,000/µL)1011.8 (9.8–22.4)515.0 (7.8–16.9)0.633
 Absolute neutrophil count (1,000/µL)109.7 (7.6–17.1)513.2 (6.7–16.7)0.859
 % Lymphocytes1014.5 (8.8–16.3)57.0 (5.5–12.0)0.239
 Platelet count (1,000/µL)10121.5 (53.5–224.8)565.0 (27.0–132.5)0.240
 Serum creatinine (mg/dL)101.7 (1.5–4.7)53.3 (1.7–7.7)0.355
 Blood urea nitrogen (mg/dL)1065.5 (36.5–97.8)568.0 (51.5–162.5)0.592
 Total serum bilirubin (mg/dL)
  Bilirubin direct65.1 (0.9–10.8)52.7 (0.8–8.2)0.459
  Bilirubin indirect61.0 (0.7–3.9)51.0 (0.5–9.0)1.000
 Serum potassium (meq/L)103.9 (3.6–4.4)34.7 (4.7–5.3)0.059
Complications
 Oliguria¶100 (0%)55 (100%)0.0003
 Pulmonary hemorrhage#100 (0%)51 (20%)0.333
 Lung injury**100 (0%)53 (75%)0.022
Outcomes
 Death100 (0%)51 (20%)0.333
 ICU admission100 (0%)52 (40%)0.095
 Mechanical ventilation100 (0%)52 (40%)0.095
 Dialysis100 (0%)54 (80%)0.004
Laboratory data
 Agglutinating antibody titers
  Acute phase101,600 (0–8,000)50 (0–3,300)0.220
  Convalescent phase93,200 (50–3,200)43,200 (3,200–5,600)0.339
Leptospira load (Geq/mL)††90.0 (0–0)45,690 (0–17,521)0.077

* Acute group did not meet the definition of the severe group.

† Severe group patients showed evidence of renal dysfunction¶ and/or lung injury** during hospitalization.

P-values determined by Fisher’s exact test for categorical variables or Mann–Whitney t-test for continuous variables; P-value < 0.05 considered significant.

§ Days of symptoms before hospital admission.

‖ Day of admission.

¶ Oliguria defined by oliguria (< 500 mL urine/day) or anuria (< 50 mL urine/day) within 24 hours of hospital admission or patient received hemodialysis.

# Pulmonary hemorrhage defined by mechanical ventilation, ≥ 250 mL blood in lungs, or endotracheal tube.

** Lung injury defined by mechanical ventilation, ≥ 250 mL blood in lungs or endotracheal tube, and/or respiration rate > 38/minutes.

†† Geometric mean of Leptospira genomes/mL as determined by RT-qPCR.

We performed assays on a BD Aria II 3-laser flow cytometer (Becton Dickinson, São Paulo, Brazil). For TLR markers, we stained fresh, heparinized whole blood on the day of collection (stored < 4 hours at room temperature [RT]) for 30 minutes with CD15-AlexaFluor647 (W6D3), TLR2-FITC (TL2.1; eBiosciences, Waltham, MA), and TLR4-PE (HTA125; eBiosciences). For activation marker quantification, we stimulated (0.05 µg Escherichia coli LPS, 0.5 µg Pam3CSK4, or 0.3 µg unmethylated CpG dinucleotides (single-stranded DNA; CpG motifs); InvivoGen, San Diego, CA) and labeled (CD15-AlexaFluor647 [W6D3], CD11b-APC-Cy7 [ICRF44], and CD62L-FITC [DREG56]) 100 µL whole blood as previously described.27 We washed with cold fluorescent-activated cell sorting (FACS) buffer (FACS buffer = phosphate buffered saline + 2% fetal bovine serum + 0.1% NaN3), lysed for 10–15 minutes in 1 mL freshly prepared 1× BD lysing solution in ddH20 (Becton Dickinson, São Paulo, Brazil), and fixed in 4% paraformaldehyde for 10 minutes at RT. We washed fixed cells with 400 µL cold FB, resuspended in 400 µL FB, and incubated at 4°C until analyzed on the flow cytometer (within 24 hours). We prepared cells, isotype controls, and compensation beads (to determine gating) for every experiment using the Becton–Dickinson flow cytometry staining support protocols. We determined the median fluorescent intensity (MFI) from 50,000 events within the granulocyte population after subtraction of isotype control levels and defined a marker’s MFI fold-change as MFI (stimulated with TLR agonists LPS, Pam3, or CpG) divided by MFI (medium only).

Precise mechanisms leading to high case fatality in leptospirosis patients are currently unknown. To assess whether the neutrophilia observed in leptospirosis patients was associated with increased neutrophil activation and relevant for multi-organ dysfunction, we characterized the neutrophil response during acute infection. We found no significant difference in the abundance of neutrophils or other circulating immune cell types between patients with (S; n = 5) or without (A; n = 10) renal/lung dysfunction (Table 1). We quantified Leptospira load (Leptospira genome/mL blood) using a standard TaqMan qPCR protocol targeting the lipL32 gene or 16S rRNA gene for amplification.25 Severe patients (S) trended higher for Leptospira loads, suggesting organ dysfunction correlated with greater pathogen burden (Table 1).

We investigated whether differences in neutrophil activation or function were associated with disease outcome by quantifying two neutrophil activation markers, CD11b (β2 integrin) and CD62L (L-selectin), in acute and convalescent samples from patients with confirmed leptospirosis.27 We detected comparable baseline frequencies (%CD15+ cells, Figure 1) and activation levels of neutrophils during acute infection (Figure 1A and B), suggesting circulating neutrophil activation does not correlate with disease severity. Unexpectedly, we observed no significant difference in the abundance of neutrophils with activation markers (CD11b and CD62L) between patients’ paired acute and convalescent samples or samples from healthy adults although the healthy controls trended higher in CD62L expression (less activated) than patients lacking organ dysfunction (A) (P = 0.07). These data suggest activated neutrophils may have migrated to sites of infection in patients and were undetectable in peripheral samples (Figure 1A and B).

Figure 1.
Figure 1.

Neutrophil activation was not associated with disease severity. Expression of CD62L (A) and f CD11b (B) on neutrophils during acute leptospirosis indicate activation levels similar to those in convalescent patients and healthy controls. Results are shown as the percent of granulocytes positive for CD15 and CD62L (A) or CD11b (B) and MFI of CD62L (A) or CD11b (B) on CD15+ granulocytic cells. Groups are A (acute leptospirosis, no renal/lung dysfunction; n = 6), S (severe acute leptospirosis, renal/lung dysfunction; n = 4), C (convalescent time point; n = 4), and H (healthy controls; n = 4). ns indicates no significant differences in group comparisons (nonparametric one-way ANOVA). Means ± SEM are shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 3; 10.4269/ajtmh.19-0160

We also hypothesized that greater disease severity could be associated with increased neutrophil functional activity, resulting in release of potentially damaging enzymes or reactive oxygen and nitrogen species. Therefore, we quantified neutrophil response to stimulation with agonists for TLR2/1 (Pam3CSK4), TLR4 (LPS), or TLR9 (CpG). Both patient groups showed similar activation levels (Figure 2, higher CD11b MFI fold-changes and lower CD62L MFI fold-changes) following stimulation, indicating no severity-associated difference in neutrophil functional response in patients with severe infection (Figure 1C and D).

Figure 2.
Figure 2.

Neutrophil activation function was not associated with disease group. Cells were stimulated with toll-like receptor agonists as described. Results are represented as the means ± SEM for MFI fold-changes of CD62L (A) and CD11b (B) on CD15+ granulocytes. A value of “1” indicates no response to agonist stimulation relative to unstimulated controls. Groups are A (acute leptospirosis, no renal/lung dysfunction; n = 6), S (severe acute leptospirosis, renal/lung dysfunction; n = 4), and C (convalescent time point; n = 4). ns indicates no significant differences in group comparisons (nonparametric one-way ANOVA). Means ± SEM are shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 3; 10.4269/ajtmh.19-0160

Most in vitro experiments on healthy human cells have shown that TLR2, not TLR4, is the primary receptor for leptospiral LPS (unlike other Gram-negative bacteria), although there are reports of whole leptospires signaling through both TLR2 and TLR4 in human cell lines, whole blood, and PBMCs.1518 To define the roles of TLR2 and TLR4 in responding to leptospires, we quantified neutrophil TLR2 and TLR4 expression during acute, paired convalescent leptospirosis samples, and in patients with other febrile diseases. We observed elevated levels of TLR2 and TLR4 in severe (S) and acute (A) leptospirosis patients compared with healthy volunteers and paired convalescent samples (Figure 3). Notably, TLR2 and TLR4 expression was significantly higher on neutrophils from patients with more severe leptospirosis.

Figure 3.
Figure 3.

Increased toll-like receptor (TLR) expression on neutrophils in acute leptospirosis patients with renal/lung dysfunction. (A) Increased TLR2 expression in patients with renal and/or lung dysfunction (group S). (B) TLR4 expression on neutrophils was lower than TLR2 expression but displayed a similar expression pattern across groups. Results are represented as the percent granulocytes positive for CD15 and TLR2 (A) or TLR4 (B) after isotype subtraction. Groups are A (acute leptospirosis, no renal/lung dysfunction; n = 10), S (severe acute leptospirosis, renal/lung dysfunction; n = 5), C (convalescent time point; n = 5), and H (healthy Brazilian adults; n = 4). Asterisks indicate significant differences relative to group S (*P < 0.05, **P < 0.01). Means ± SEM are shown. No other significant differences between groups were identified.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 3; 10.4269/ajtmh.19-0160

To identify possible mechanisms relevant to disease severity, we compared TLR expression with clinical characteristics (Supplemental Tables 1 and 2), but found no association among neutrophil TLR expression, days of symptoms, or percent neutrophils at the time of hospital admission. However, higher bacterial loads in whole blood correlated independently with higher expression of both TLR2 and TLR4 (Pearson’s correlation, TLR2: r = 0.810 [0.468–0.941], P = 0.0008 and TLR4: r = 0.636 [0.131–0.879], P = 0.019).

We hypothesize that higher bacterial loads from larger inocula are a key factor driving disease pathology, with the increased bacteria loads leading to elevated TLR responses. Neutrophils may have reduced activity against Leptospira,13,24,28 and we recently demonstrated low expression of the neutrophil antimicrobial peptide, cathelicidin, correlated with fatal leptospirosis cases, indicating a role for effective bactericidal response.29 Protection from Leptospira infection is likely mediated in part through anti-leptospiral LPS antibodies,15 the production of which could be affected by neutrophil function, and our previous results showed significant decreases in antibody production in fatal cases.29 These results may indicate that the elevated levels of neutrophils are associated with more severe disease11,12 which is due to reduced migration to sites of infection rather than aberrant activation. However, in cases of fatal human leptospirosis, autopsies have found neutrophils and other immune cells in multiple organs, although not the lungs, indicating possible direct involvement in the pathology of severe disease.30,31 Other support for neutrophil involvement includes modulation of T-cell, natural killer, and B-cell responses through cytokine and chemokine production (IL-10 and IL-6), which are elevated in fatal cases of leptospirosis.32,33 Thus, there is still much to learn about the role of neutrophils in the pathogenesis of leptospirosis.

In summary, this is a preliminary report describing responses of neutrophils during acute leptospirosis. We found that neutrophils expressed significantly higher TLR2 in patients with renal and/or lung dysfunction relative to patients lacking organ failure, and that TLR2 and TLR4 expression levels correlate with bacterial loads. This is consistent with a report demonstrating neutrophils expressed TLR2 during acute leptospirosis,13 but in contrast to a lack of increased neutrophil activation among patients observed by Raffay et al.13 These contrasts could be due to differences in study populations and infecting Leptospira serovars. Future longitudinal analyses of neutrophil and macrophage function during the course of illness in a larger patient cohort and of cathelicidin, which has immunomodulatory and bactericidal activities, may distinguish critical contributions of innate immune cells to the resolution of human Leptospira infections and severe disease outcomes.

Supplemental materials

Acknowledgments:

We would like to thank the patients and families for their participation, the medical workers at the Hospital Couto Maia, the Flow Cytometry Platform at Fiocruz-Bahia, and the entire Leptospirosis Team at Fiocruz-Bahia for their continuous support and collaboration. This work was supported by funding from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health [grant number U01AI088752 to A. I. K.; AI 089992 to R. R. M. and A. C. S.; and AG 042489 to A. C. S.]; an American Society for Tropical Medicine and Hygiene Gorgas Memorial Institute Research Award to J. C. L.; a Fogarty International Center, Global Health Equity Scholars Fellowship [grant number R25 TW009338 to J. C. L.]; and a Fundação Oswaldo Cruz/Conselho Nacional de Desenvolvimento Científico e Tecnológico–Ciência Sem Fronteiras Bolsa Jovens Talentos to J. C. L.

REFERENCES

  • 1.

    Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-Ridder B, Ko AI, 2015. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 9: e0003898.

    • Search Google Scholar
    • Export Citation
  • 2.

    Ko AI, Goarant C, Picardeau M, 2009. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7: 736747.

    • Search Google Scholar
    • Export Citation
  • 3.

    Gouveia EL, Metcalfe J, de Carvalho AL, Aires TS, Villasboas-Bisneto JC, Queirroz A, Santos AC, Salgado K, Reis MG, Ko AI, 2008. Leptospirosis-associated severe pulmonary hemorrhagic syndrome, Salvador, Brazil. Emerg Infect Dis 14: 505508.

    • Search Google Scholar
    • Export Citation
  • 4.

    Vieira SR, Brauner JS, 2002. Leptospirosis as a cause of acute respiratory failure: clinical features and outcome in 35 critical care patients. Braz J Infect Dis 6: 135139.

    • Search Google Scholar
    • Export Citation
  • 5.

    Marotto PC, Nascimento CM, Eluf-Neto J, Marotto MS, Andrade L, Sztajnbok J, Seguro AC, 1999. Acute lung injury in leptospirosis: clinical and laboratory features, outcome, and factors associated with mortality. Clin Infect Dis 29: 15611563.

    • Search Google Scholar
    • Export Citation
  • 6.

    Murgia R, Garcia R, Cinco M, 2002. Leptospires are killed in vitro by both oxygen-dependent and independent reactions. Infect Immun 70: 71727175.

    • Search Google Scholar
    • Export Citation
  • 7.

    Linde A, Lushington GH, Abello J, Melgarejo T, 2013. Clinical relevance of cathelicidin in infectious disease. J Clin Cell Immunol S13: 003.

  • 8.

    Scharrig E, Carestia A, Ferrer MF, Cedola M, Pretre G, Drut R, Picardeau M, Schattner M, Gomez RM, 2015. Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PLoS Negl Trop Dis 9: e0003927.

    • Search Google Scholar
    • Export Citation
  • 9.

    Pillai PS et al. 2016. Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease. Science 352: 463466.

  • 10.

    De Silva NL, Niloofa M, Fernando N, Karunanayake L, Rodrigo C, De Silva HJ, Premawansa S, Handunnetti SM, Rajapakse S, 2014. Changes in full blood count parameters in leptospirosis: a prospective study. Int Arch Med 7: 31.

    • Search Google Scholar
    • Export Citation
  • 11.

    Craig SB, Collet TA, Wynwood SJ, Smythe LD, Weier SL, McKay DB, 2013. Neutrophil counts in leptospirosis patients infected with different serovars. Trop Biomed 30: 579583.

    • Search Google Scholar
    • Export Citation
  • 12.

    Craig SB, Graham GC, Burns MA, Dohnt MF, Smythe LD, McKay DB, 2009. Haematological and clinical-chemistry markers in patients presenting with leptospirosis: a comparison of the findings from uncomplicated cases with those seen in the severe disease. Ann Trop Med Parasitol 103: 333341.

    • Search Google Scholar
    • Export Citation
  • 13.

    Raffray L, Giry C, Vandroux D, Kuli B, Randrianjohany A, Pequin AM, Renou F, Jaffar-Bandjee MC, Gasque P, 2016. Major neutrophilia observed in acute phase of human leptospirosis is not associated with increased expression of granulocyte cell activation markers. PLoS One 11: e0165716.

    • Search Google Scholar
    • Export Citation
  • 14.

    Futosi K, Fodor S, Mocsai A, 2013. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol 17: 638650.

    • Search Google Scholar
    • Export Citation
  • 15.

    Zuerner RL, 2015. Host response to Leptospira infection. Curr Top Microbiol Immunol 387: 223250.

  • 16.

    Nahori MA, Fournie-Amazouz E, Que-Gewirth NS, Balloy V, Chignard M, Raetz CR, Saint Girons I, Werts C, 2005. Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells. J Immunol 175: 60226031.

    • Search Google Scholar
    • Export Citation
  • 17.

    Werts C et al. 2001. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat Immunol 2: 346352.

  • 18.

    Goris MG, Wagenaar JF, Hartskeerl RA, van Gorp EC, Schuller S, Monahan AM, Nally JE, van der Poll T, van’t Veer C, 2011. Potent innate immune response to pathogenic Leptospira in human whole blood. PLoS One 6: e18279.

    • Search Google Scholar
    • Export Citation
  • 19.

    Viriyakosol S, Matthias MA, Swancutt MA, Kirkland TN, Vinetz JM, 2006. Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden. Infect Immun 74: 887895.

    • Search Google Scholar
    • Export Citation
  • 20.

    Chassin C et al. 2009. TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans. J Immunol 183: 26692677.

    • Search Google Scholar
    • Export Citation
  • 21.

    Guo Y, Fukuda T, Donai K, Kuroda K, Masuda M, Nakamura S, Yoneyama H, Isogai E, 2015. Leptospiral lipopolysaccharide stimulates the expression of toll-like receptor 2 and cytokines in pig fibroblasts. Anim Sci J 86: 238244.

    • Search Google Scholar
    • Export Citation
  • 22.

    Guo Y, Fukuda T, Nakamura S, Bai L, Xu J, Kuroda K, Tomioka R, Yoneyama H, Isogai E, 2015. Interaction between leptospiral lipopolysaccharide and toll-like receptor 2 in pig fibroblast cell line, and inhibitory effect of antibody against leptospiral lipopolysaccharide on interaction. Asian-Australas J Anim Sci 28: 273279.

    • Search Google Scholar
    • Export Citation
  • 23.

    Zhang W, Zhang N, Xie X, Guo J, Jin X, Xue F, Ding Z, Cao Y, 2016. Toll-like receptor 2 agonist Pam3CSK4 alleviates the pathology of leptospirosis in hamster. Infect Immun 84: 33503357.

    • Search Google Scholar
    • Export Citation
  • 24.

    Chen X, Li SJ, Ojcius DM, Sun AH, Hu WL, Lin X, Yan J, 2017. Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis. PLoS One 12: e0181014.

    • Search Google Scholar
    • Export Citation
  • 25.

    Stoddard RA, Gee JE, Wilkins PP, McCaustland K, Hoffmaster AR, 2009. Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene. Diagn Microbiol Infect Dis 64: 247255.

    • Search Google Scholar
    • Export Citation
  • 26.

    Smythe LD, Smith IL, Smith GA, Dohnt MF, Symonds ML, Barnett LJ, McKay DB, 2002. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infect Dis 2: 13.

    • Search Google Scholar
    • Export Citation
  • 27.

    Qian F, Guo X, Wang X, Yuan X, Chen S, Malawista SE, Bockenstedt LK, Allore HG, Montgomery RR, 2014. Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging. Aging (Albany NY) 6: 131139.

    • Search Google Scholar
    • Export Citation
  • 28.

    Wang B, Sullivan J, Sullivan GW, Mandell GL, 1984. Interaction of leptospires with human polymorphonuclear neutrophils. Infect Immun 44: 459464.

    • Search Google Scholar
    • Export Citation
  • 29.

    Lindow JC et al. 2016. Cathelicidin insufficiency in patients with fatal leptospirosis. PLoS Pathog 12: e1005943.

  • 30.

    Arean VM, 1962. The pathologic anatomy and pathogenesis of fatal human leptospirosis (Weil’s disease). Am J Pathol 40: 393423.

  • 31.

    Sitprija V, Evans H, 1970. The kidney in human leptospirosis. Am J Med 49: 780788.

  • 32.

    Jaillon S, Galdiero MR, Del Prete D, Cassatella MA, Garlanda C, Mantovani A, 2013. Neutrophils in innate and adaptive immunity. Semin Immunopathol 35: 377394.

    • Search Google Scholar
    • Export Citation
  • 33.

    Reis EA, Hagan JE, Ribeiro GS, Teixeira-Carvalho A, Martins-Filho OA, Montgomery RR, Shaw AC, Ko AI, Reis MG, 2013. Cytokine response signatures in disease progression and development of severe clinical outcomes for leptospirosis. PLoS Negl Trop Dis 7: e2457.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Janet C. Lindow, Biomedical Research and Education Foundation of Southern Arizona, 3601 S. 6th Ave. Bldg. 77, MC (0-151), Tucson, AZ 85723. E-mail: jlindow@alum.mit.edu

Authors’ addresses: Janet C. Lindow, Department of Psychiatry, University of Arizona, the Biomedical Research and Education Foundation of Southern Arizona, Tucson, AZ, and Southern Arizona Virginia Health Care System, Tucson, AZ, E-mail: jlindow@alum.mit.edu. Annie J. Tsay, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, E-mail: tsayorama@gmail.com. Ruth R. Montgomery, Section of Rheumatology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, E-mail: ruth.montgomery@yale.edu. Eliana A. G. Reis and Nivison R. R. Nery Jr., Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil, E-mails: eagreis@gmail.com and nivison@conveniado.bahia.fiocruz.br. Elsio A. Wunder Jr. and Albert I. Ko, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, and Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil, E-mails: elsio.wunder@yale.edu and albert.ko@yale.edu. Guilherme Araújo, Universidad Privada del Este, Ciudad del Este, Paraguay, E-mail: guyaraujo@gmail.com. Subhasis Mohanty and Albert C. Shaw, Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, E-mails: subhasis.mohanty@yale.edu and albert.shaw@yale.edu. Patty J. Lee, Division of Pulmonary, Allergy & Critical Care, Duke University Medical Center, Durham, NC, E-mail: patty.lee@duke.edu Mitermayer G. Reis, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil, and Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Praça Conselheiro Almeida Couto, Largo do Terreiro de Jesus, Salvador, Bahia, E-mail: miter@bahia.fiocruz.br.

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