Elevated Soluble CD163 Plasma Levels Are Associated With Disease Severity In Patients With Hemorrhagic Fever With Renal Syndrome

Hemorrhagic fever with renal syndrome(HFRS), resulting from Hantavirus infection, is one of the serious zoonotic diseases with fever, Hypotension shock, hemorrhage and renal failure are regarded as the main clinical performance. There are five successive clinical stages in typical HFRS patients. These include the fever, hypotensive shock, oliguric, polyuric and convalescent stage. Currently, there are as many as 100,000 cases of HFRS reported annually throughout the world, of which greater than 70%~90% were documented in China, with a mortality rate of 1% ~ 10%. The most remarkably pathological characteristics of HFRS are endothelial inflammation, loss of endothelial barrier function, immune cell migration and increased vascular permeability. However, the pathogenesis of HFRS has not been entirely elucidated, and it has been generally considered that viral replication and with the immune response, which including platelet dysfunction, immune complexes formation, B cell and T cell response, complement activation and HTNV-induced cytokine and chemokines production, are participate in tissue injury.Monocytes and macrophages constitute a significant component of the immune responses against viruses. These cells trigger inflammation as well as bridging innate and adaptive immunity following viral infections. Monocytes in circulation represent a heterogeneous population. Three major subsets of monocytes have been identified based upon the relative expression of CD14 and CD16. Classical monocytes(CD14++CD16-, Mon1) account for 80%~90% of the monocytes in circulation, whereas the intermediate(CD14++CD16+, Mon2) and non-classical(CD14+CD16++, Mon3) monocyte subsets account for 10%~20% of the circulating monocytes. The relative monocyte distribution that is observed in circulation is dynamic and changeable as a function of the inflammatory and metabolic drivers that impact the differentiation between the subsets and their functions.The CD163 is a specific scavenger receptor for hemoglobin/heme in vivo. It located on the extracellular side of the monocytes and macrophages membrane. One of its primary and well-described functions is the clearance of extracellular hemoglobin by a means of hemoglobin–haptoglobin complex endocytosis, which thus avoids the oxidative stress associated with free hemoglobin by liberating free iron, bilirubin, and carbon monoxide. The CD163 could shed from the surface of monocyte into blood and become s CD163. The s CD163 has the effect of anti-inflammatory and immunomodulation.Increased s CD163 serum concentrations have been reported in patients who suffer from various infectious and inflammatory diseases, such as sepsis, tuberculosis, liver disease, acquired immune deficiency syndrome diabetes, rheumatoid, dengue fever and arthritis. However, it has not been investigated about the CD163 expression levels on monocyte subsets and the levels of plasma s CD163 in HFRS patients. Because of the important role of CD163 and s CD163 in immune response, we speculated that CD163 and s CD163 may have significant changes in patients with HFRS and they may play an important role in the pathogenesis of HFRS. The aims of this study were to observe the CD163 expression levels on monocyte subsets and the plasma s CD163 levels in HFRS patients and to further analyze the correlation among CD163, s CD163 and disease severity. Patients and MethodsPeripheral blood mononuclear cell(PBMC) and plasma samples were collected from 81 hospitalized patients with HFRS in Tangdu Hospital from October 2011 to January 2014 and from 15 healthy controls. First, the plasma s CD163 levels in different disease phase or in different severities of HFRS patients were measured using a sandwich ELISA. The correlations between s CD163 and disease course, disease severity-related clinical parameters were also analyzed. Furthermore, monocytes subsets were identified and CD163 expression in 3 monocytes subsets was analyzed by flow cytometry. Results 1. Changes of the soluble CD163 plasma level in the HFRS patients.The median(IQR) of plasma s CD163 level in patient with HFRS for febrile/hypotensive, oliguric, diuretic, convalescent stage, as well as in the normal controls was 3.75(2.65-6.46) mg/L, 3.58(2.17-4.28) mg/L, 2.43(1.69-3.09) mg/L, 1.80(1.43-2.38) mg/L, and 0.87(0.66-0.99) mg/L, respectively. The increased plasma s CD163 level was observed in acute stage(comprised febrile, hypotensive, and oliguric stage) when compared with normal controls(P <0.0001), and the decreased in convalescent phase(comprised diuretic or convalescent stage), but still higher than that of normal controls(P <0.0001). The plasma s CD163 levels in the patients with different disease severities displayed a similar change trend. However, a more obvious decline was observed in the severe/critical patient group. The s CD163 plasma levels in the severe/critical group were higher than those in the mild/moderate group during the acute, only 4(13.7%) of the 29 mild/moderate group cases had plasma s CD163 levels that were over 4 mg/L, while 28(60.8%) of the 46 severe/critical group cases had s CD163 levels that were over 4 mg/L(a 4.3-fold change between the high vs. mild/moderate groups). Furthermore, the relationships between plasma s CD163 levels and clinical parameters that could represent the severity of the disease and inflammatory cytokine levels were analyzed. The result revealed a negative correlation between plasma s CD163 levels and the decreased platelet count(r=-0.6109, P <0.0001), and positive correlations between plasma s CD163 levels and the increased white blood cell count(r =0.5322, P <0.0001), or the increased level of serum creatinine(r =0.3718, P <0.0001), or the increased level of blood urea nitrogen(r =0.38, P <0.0001), or the increased plasma level of IL-6(r= 0.4837, P <0.0001), or the increased plasma level of IFN-γ( r= 0.4929, P <0.0001). 2. Changes of monocyte subsets in patients with HFRSClassical(CD14++CD16-), intermediate(CD14++CD16+) and non-classical monocytes(CD14+CD16++) subsets were identified by flow cytometry based on their forward and side scatter characteristics and by their CD14 and CD16 expression levels. The results showed the intermediate(CD14++CD16+) monocyte ratio increased significantly in the acute phase compared with normal controls(P <0.0001). However, the classical(CD14++CD16-) and non-classical(CD14+CD16++) monocyte ratios were reduced compared with normal controls(P <0.0001). Though the CD163 expression on the three monocytes subsets was increased during the acute stage, the CD163 expression on the intermediate(CD14++CD16+) monocytes was the highest levels when compared with the CD163 expression on the other monocyte subsets(P <0.0001). Additionally, the concentration of plasma s CD163 correlated with expansion of the intermediate(CD14++CD16+) monocytes(r =0.5779, P < 0.0001) and increased CD163 expression on intermediate(CD14++CD16+)monocyte(r = 0.6245, P < 0.0001). ConclusionIn summary,the elevated s CD163 plasma level seemed to be a feature in HFRS patients. The monocyte subset proportions were altered in the patients with HFRS and CD163 was differentially expressed on the monocyte subsets. These changes are the most apparent in the acute phase. Levels of plasma s CD163 is closely related to clinical parameters that could represent the severity of the disease and is closely related to inflammatory cytokine levels that could represent the extent of the body’s inflammatory response. These results suggest that s CD163 may be correlated with disease severity and the disease progression in HFRS patients and s CD163 is a novel marker for HFRS. Forthermore, the results are useful for understanding the monocyte-mediated disease progression, monocyte-mediated immunity and immune homeostasis after HTNV infetion in human.