Biomarkers for predicting the outcome of Puumala hantavirus infection

Abstract

Hantaviruksiin kuuluva Puumala-virus aiheuttaa lievän munuaisoireisen verenvuotokuumeen, myyräkuumeen. Taudin vaikeusaste vaihtelee oireettomasta kuolemaan johtaviin tapauksiin, jotka ovat kuitenkin harvinaisia. Myyräkuumeen taudin kehittymistä ei täysin tunneta, mutta immunologisilla reaktioilla on arveltu olevan siinä tärkeä osuus. Väitöstutkimuksessa selvitettiin erilaisten immunologisten tekijöiden yhteyttä myyräkuumeen vaikeusasteeseen sekä arvioitiin niiden mahdollista osuutta taudin kehittymisessä.

Korkean IL-6-pitoisuuden todettiin liittyvän vaikeaan myyräkuumeeseen ja olevan itsenäinen riskitekijä munuaisten vajaatoiminnalle.

Pitkän pentraksiinin PTX3:n pitoisuus oli koholla akuutissa myyräkuumeessa ja korkea pitoisuus oli yhteydessä vaikeaan taudinkuvaan, erityisesti matalaan verihiutaletasoon sekä myös komplementtijärjestelmän aktivaatioon. Tutkimuksen perusteella PTX3 voisi vaikuttaa matalan trombosyyttitason kehittymiseen myyräkuumeessa aktivoituneen komplementtijärjestelmän kautta.

Tryptofaania kataboloivan entsyymin IDO:n pitoisuuden todettiin olevan koholla akuutissa myyräkuumeessa ja korkea IDO oli yhteydessä vaikeaan tautiin sekä osoittautui merkittävän munuaisten vajaatoiminnan itsenäiseksi riskitekijäksi. IDO saattaa vaikuttaa munuaisten vajaatoiminnan kehittymiseen myyräkuumepotilailla aiheuttamalla tubulaaristen epiteelisolujen apoptoosia tai lamaamalla immuunijärjestelmää T-soluja estämällä.

Soluvapaa DNA on peräisin apoptoottisista tai nekroottisista soluista ja siten kuvastaa solutuhon määrää erilaisissa sairauksissa. Plasman soluvapaan DNA:n pitoisuus todettiin koholla olevaksi akuutissa myyräkuumeessa. Se myös korreloi taudin vaikeuteen mm. sairaalahoidon kestolla arvioituna. Virtsan soluvapaan DNA:n eritys puolestaan ei ollut myyräkuumeessa koholla eikä se korreloinut taudin vaikeusasteeseen. Siten virtsan soluvapaan DNA:n eritys ei kuvasta tulehduksen määrää munuaisissa.

Korkea plasman CRP ei liittynyt vaikeampaan akuuttiin myyräkuumeeseen vaan päinvastoin osoittautui mahdolliseksi munuaisten toimintaa suojaavaksi tekijäksi. CRP ei siten sovellu seurantamerkkiaineeksi arvioitaessa taudin vaikeusastetta ja ennustetta myyräkuumepotilailla.

Puumala hantavirus (PUUV) causes a mild type of hemorrhagic fever with renal syndrome called nephropathia epidemica (NE). After an incubation period of 1-8 weeks, NE presents with sudden high fever, headache, nausea, abdominal pain, backache, visual disturbances, and impaired renal function. The severity of NE varies from asymptomatic to rare fatal cases, and its pathogenesis is not completely understood. An important feature in hantaviral infections is capillary leakage due to increased capillary permeability. The mechanisms behind this phenomenon are unclear, although immunological responses have been suggested to be important.

In the present study, the association of immunological factors, i.e. interleukin (IL)-6, C-reactive protein (CRP), pentraxin-3 (PTX3), indoleamine 2,3-dioxygenase (IDO), and cell-free DNA (cf-DNA), with the severity of NE was analyzed. Furthermore, their possible role in the pathogenesis was assessed.

Pentraxins are a family of acute-phase proteins. CRP is a short pentraxin mainly produced in the liver in response to inflammatory signals. IL-6, in turn, is a multifunctional cytokine involved in immune responses and inflammation. Increased cytokine levels have previously been found in the plasma, urine, and tissues of patients with hantavirus infection. In Study I, plasma IL-6 and CRP as well as their association with disease severity reflecting variables were studied in 118 hospital-treated patients with acute NE. High plasma IL-6 levels were found to associate with clinically severe acute NE. High IL-6 levels were also found as an independent risk factor for impaired renal function. High plasma CRP, in turn, did not have an association with a more severe course of the disease. On the contrary, high CRP levels turned out to be a possible protective factor for renal function.

PTX3 is a long pentraxin produced at the site of inflammation. In Study II, 61 hospitalized PUUV-infected patients were studied to assess the associations of plasma PTX3 with variables reflecting the severity of acute NE. PTX3 levels were shown to be elevated during the acute phase of the disease. High PTX3 associated with a more severe course of NE and, most of all, with significant thrombocytopenia. It also associated with the activation of the complement system. Thus, PTX3 could possibly be involved in the pathogenesis of thrombocytopenia in NE through the activated complement cascade.

IDO is the rate-limiting enzyme in tryptophan catabolism to kynurenine leading to depletion of tryptophan as well as T cell suppression. In Study III, 102 hospitalized patients were studied to establish the association of serum IDO enzyme with the variables reflecting the severity of acute NE. Serum tryptophan and kynurenine levels were determined by reverse-phase high-performance liquid chromatography, and tryptophan/kynurenine ratio reflecting IDO activity was calculated. IDO levels were found to be elevated during acute NE. High IDO was revealed to associate with clinically severe NE and it presented as an independent risk factor for significant renal insufficiency. Furthermore, serum IDO levels were shown to peak before serum creatinine levels. It is conceivable that IDO is involved in the pathogenesis of renal insufficiency in PUUV infection. The possible mechanisms are promotion of tubular epithelial cell apoptosis or immunosuppression through T cell suppression.

Elevated levels of cf-DNA have been previously reported in different clinical disorders. The current view is that, in these conditions, cf-DNA originates from apoptotic or necrotic cells and therefore reflects the amount of cellular damage. In Study IV, total cf-DNA was studied in the plasma of 61 patients and urine of 20 patients with acute NE. Also, a qualitative high-sensitivity lab-on-a-chip DNA assay was carried out in 20 patients to elucidate the appearance of cf-DNA in plasma and urine. The plasma levels of cf-DNA were found to be elevated during acute PUUV infection and correlate with the apoptotic band (150-200 base pairs) intensity. The total plasma cf-DNA concentration also correlated with leukocytosis, thrombocytopenia, and the length of hospitalization. The urinary excretion of cf-DNA, in turn, was not elevated during the acute infection and it did not correlate with any of the disease severity reflecting variables. In conclusion, high plasma IL-6, PTX3, cf-DNA, and serum IDO levels reflect the clinical severity of NE, while high CRP concentration seems to protect against renal failure and does not predict a severe course of NE. Neither does urinary excretion of cf-DNA reflect the degree of inflammation in the kidney. Furthermore, PTX3 might be involved in the pathogenesis of thrombocytopenia and IDO, in turn, could act in the pathogenesis of renal insufficiency.