Relationship between Anaemia, Haemolysis, Inflammation and Haem Oxygenase-1 at Admission with Sepsis: a pilot study

Upregulation of haem oxygenase-1 (HO-1), due to haemolysis and/or inflammation, can lead to impaired immune function. Anaemia is common among sepsis patients, but the consequences of sepsis-associated anaemia are poorly understood. Here, our objective was to determine the prevalence and extent of anaemia, haemolysis, inflammation, and HO-1 induction after early hospital admission. We hypothesised that inflammation- or infection-induced haemolysis contributes to sepsis-associated anaemia and that this will lead to expression of HO-1. In this study, plasma obtained from seventy adult patients within 12 hours of admission to intensive care due to sepsis were analysed for anaemia, haemolysis and inflammatory markers by ELISA and microbead array. The majority (82.6%) of patients were anaemic with evidence of haemolysis (raised haem, haptoglobin, haemopexin, and HO-1 concentrations). Interestingly, concentrations of both haemoglobin and IL-10 were moderately positively correlated with HO-1 concentration (Hb: r = 0.32, p = 0.007; IL-10 r = 0.39, p = 0.0008) whereas HO-1 concentration was weakly negatively correlated with haemopexin (r = −0.23, p = 0.055). Anaemia, while common, was not associated with HO-1 concentration. After adjusting for confounding, HO-1 induction appears to be associated primarily with IL-10 concentration rather than haemolysis. Disease severity at diagnosis was correlated with early plasma IL-10 (r = 0.35, p = 0.003) and HO-1 (r = 0.24, p = 0.048) concentrations. Notably, admission levels of haem, HO-1, and IL-10 were indicators of survival.

SCIENTIFIC REPoRtS | (2018) 8:11198 | DOI: 10.1038/s41598-018-29558-5 HO-1 induction has been reported in liver and in blood monocytes of sepsis patients 17,18 . In humans with malaria-induced haemolysis, raised plasma HO-1 concentrations correlate with severely impaired neutrophil respiratory burst 19,20 and, in mice with haemolysis, neutrophil function can be restored by specific inhibition of HO-1 activity 19 suggesting a role for HO-1 in increased susceptibility to invasive bacterial disease 19,21 . However, HO-1 concentrations also correlate with the acute phase response (C reactive protein) 19 indicating a role for host inflammatory responses in initiating or potentiating HO-1 induction. These synergistic interactions between anaemia, infection, inflammation and HO-1 may be occurring in sepsis patients. For example, in human sepsis, haemoglobin concentrations decrease in a hepcidin-dependent manner during admission, hepcidin and IL-6 concentrations are positively correlated and hepcidin concentrations are highest in those with the most severe disease 22 . Moreover, free haemoglobin is associated with reduced survival in sepsis 23 and higher concentrations of haptoglobin and haemopexin have been observed in survivors than among those who died, indicating that haemolysis may be occurring in sepsis, contributing to anaemia 16,24 .
In this context, we hypothesized that haemolysis may be contributing to anaemia in critically ill sepsis patients and may result in induction of HO-1, which may synergize with inflammation to impair innate immune function. In this pilot study, hospital records and plasma were analysed from samples collected within 12 hours of ICU admission of sepsis patients whom did not have any documented immune comorbidity 25 . The aims of the study were to determine the prevalence of, and the interaction between, anaemia, haemolysis, inflammation and disease severity and particularly, to understand its relationship to HO-1 induction at the time of admission to the ICU for bacterial sepsis. These data provide a secure foundation for future prospective studies of the relationship between sepsis, haemolysis and immune function.
Sepsis patients are mildly to moderately anaemic at admission. Median haematocrit, red cell count and haemoglobin concentration were all below the healthy range in both male and female sepsis patients ( Fig. 1A-C, Table S2). According to WHO guidelines 28 , 83% of patients were clinically anaemic at admission (Hb < 13 g/dL in males, Hb < 12 g/dL in females) (Fig. 1D), with most being moderately anaemic, i.e., with haemoglobin concentrations in the range 8-10.9 g/dL (Fig. 1E). Total haemoglobin (Hb) concentration was highly correlated with erythrocyte count (r = 0.84, p < 0.0001) ( Fig. 2A). Mean corpuscular volume (MCV) was within the normal range for most patients (Fig. S1F).
Sepsis and HO-1. As HO-1 can be induced by haemolysis and, independently, by cytokines (in particular, interleukin (IL)-10 29-32 ), we next determined the levels of plasma HO-1 and whether haemolysis (defined by haem and haemopexin levels) or IL-10 was the most likely driver of HO-1 induction.

Relationship between HO-1 induction and cytokine concentrations in sepsis patients. As
HO-1-mediated catabolism of haem to carbon monoxide is a major pathway for the anti-inflammatory properties of IL-10 29 , we measured concentrations of IL-10, tumour necrosis factor alpha (TNFα), granulocyte-colony stimulating factor (G-CSF), and IL-6 (Figs 3A-D and S2). IL-6 was significantly (positively) correlated with IL-10 concentration (r = 0.58, p < 0.0001) (Fig. 3B). None of the cytokines measured correlated with either anaemia (Hb) or haem concentrations (Fig. S3), but all showed significant, albeit weak, inverse correlations with haemopexin concentration (Figs 3C,D and S2). IL-10 was the only cytokine to be moderately and significantly correlated with HO-1 concentration (r = 0.39, p = 0.0008) (Figs 3E,F and S2). After adjusting for haemolysis (defined by both haem and HPX concentrations), IL-10 remained significantly associated with HO-1 (Table 1) whereas after adjusting for IL-10 concentration, neither haem nor HPX became or remained significantly associated with HO-1. Thus, whilst haemolysis (as measured by low HPX, reflecting cumulative exposure to haem) and IL-10 are both associated with markers of inflammation (IL-6, TNFα, and G-CSF), IL-10 is most closely associated with raised HO-1 in sepsis patients at the time of admission. can be induced by haemolysis and (in response to inflammation) by IL-10 and simultaneously increases tolerance (resilience) to infection (by detoxifying haem and producing tissue-protective carbon monoxide) 35,36 whilst reducing resistance to infection by impairing immune function 19,37 . However, inflammation, and its subsequent regulation by IL-10, is a strong predictor of mortality in sepsis 38 and IL-10 concentration is highly correlated with disease severity as measured, for example, by the APACHE II score [39][40][41] , suggesting that the immune suppressive effects of HO-1 may outweigh its tissue protective effects. To test this hypothesis, we looked for associations between disease severity (APACHE II and SOFA scores), death, haemolysis, inflammation and HO-1 (Fig. 4).
There was no association between APACHE II score at admission and any of the markers of haemolysis ( Fig. 4A-D). However, APACHE II score was significantly positively associated with both IL-10 and HO-1 concentrations ( Fig. 4E,F). Further, both IL-10 and HO-1 concentrations were both moderately, but statistically significantly, positively correlated with SOFA (Sequential Organ Failure Assessment) scores (IL10; r = 0.41, p = 0.0004, and HO-1; r = 0.38, p = 0.0009) (Fig. 4G,H). After adjusting for potential confounding, IL-10 was associated with APACHE II score but HO-1 was not (Table 1). Finally, we sought to characterize in-hospital mortality according to high or low (above or below median) concentrations of each analyte at admission. While neither anaemia nor HPX were significantly associated with mortality ( Fig. S4, Table 2), high levels of plasma haem, IL-10 and HO-1 at admission were significantly associated with risk of dying ( Fig. 4I-K, Table 2).

Discussion
The key findings of this preliminary study are that; (1) the majority of sepsis patients are moderately anaemic at admission to ICU, (2) plasma concentrations of HO-1, the inducible isoform of haem oxygenase, are markedly raised in sepsis, (3) IL-10 concentrations, rather than haemolysis, correlate most closely with HO-1, and (4) high HO-1 and IL-10 concentrations, but not anaemia, at admission correlate with disease severity (APACHE II score) and mortality.
The primary function of HO-1 is to degrade haem, which is highly pro-oxidant and cytotoxic. Thus, HO-1 is tissue protective in inflammatory situations, reducing damage from free haem but also actively protecting tissues through the actions of the haem breakdown product carbon monoxide 15 . The essential role of HO-1 is illustrated by a case of human HO-1 deficiency which led to death in childhood accompanied by anaemia, intravascular haemolysis, leucocytosis and chronic inflammation 42 . Conversely, low-dose carbon monoxide therapy is beneficial in animal models of sepsis yet has failed in one study of experimental endotoxaemia in humans 43,44 . Importantly, while HO-1-mediated degradation of haem to carbon monoxide has been proposed as a major cell , and (H) stratified by gender and classification of severity using WHO recommendations (males: non-anaemic (>13 g/dL), mild (11-12.9 g/dL), moderate (8-10.9 g/dL), and severe (<8 g/dL). For females: non-anaemic (>12 g/dL), mild (11-11.9 g/dL), moderate (8-10.9 g/dL), and severe (<8 g/dL). Data collected from complete blood count (CBC) data. Dot plots show individual patient parameters. Black lines represent medians. Shaded areas represent healthy reference ranges (see Table S2). Sepsis patients, n = 70; haemoglobin data for 1 patient was missing. stress indicator 45 , the immunoregulatory cytokine IL-10 can also induce expression of the HO-1 gene, HMOX-1 29 . Similarly to IL-10 46 , HO-1 has also been linked to immune dysfunction and loss of resistance to infection 47 . These conflicting (tissue protective but immunosuppressive) actions of HO-1 and its differing modes of induction (haemolysis and inflammation) make it difficult to discern the risks and benefits of HO-1 induction during acute infection and to ensure an optimal balance between enhanced resilience and loss of resistance. While anaemia, haemolysis and inflammation have all been described in sepsis patients 16,23,24,39,40,48,49 , the relationship between these processes and their collective relationship to disease severity is much less clearly established. Here, we sought to explore these parameters in a single patient cohort and to extend these analyses to their impact on the HO-1 pathway. Our patients had a median HPX that was markedly lower than the normal healthy range, consistent with depletion of plasma HPX due to scavenging of haem-haemopexin complexes. Three previous studies have found that patients who did not survive sepsis had lower levels of HPX than survivors 16,24,48 .
Here, however, we compared survival in those with low and high HPX and found no association (Fig. S4B). In our cohort, those with the lowest HPX concentrations had the highest concentrations of HO-1 (consistent with haemolysis leading to liberation of free haem and induction of HO-1 and scavenging of haem-haemopexin complexes) but these patients were not necessarily the most anaemic. This may reflect that these patients are iron sufficient and able to compensate for modest levels of haemolysis by de novo red blood cell production. While high haem concentrations were associated by with reduced survival in hospital, low HPX at admission did not (Fig. S4).
Our data suggest that, in sepsis, maintenance of homeostasis in the face of overwhelming inflammation 49 is the primary driver of the HO-1 pathway and that the hemolysis pathway of HO-1 induction is less important. Although the majority of patients in this study were anaemic, haemolysis may not be the sole cause of the anaemia. Anaemia of chronic disease (as suggested by high serum IL-6) may also contribute to the moderate anaemia seen in our cohort; hepcidin measurements would help to clarify this. Nevertheless, HO-1 concentration was moderately and significantly correlated with plasma IL-10 concentration and IL-10, in turn, reflected high concentrations of circulating inflammatory cytokines.
Despite the limitation that measurements were made only on the day of admission, the most parsimonious interpretation of these data (Fig. 5) is that inflammation drives a homeostatic regulatory response (mediated by IL-10), that this in turn induces transcription and translation of HO-1, and that HO-1 is the most proximal driver (of the parameters we have measured) of death in hospital. Haemolysis may exacerbate or prolong this response. However, this survival analysis does not account for other potential confounders, changes in the measured parameters over time, or readmission after discharge. One hypothesis to be explored in future, longitudinal and multicentre studies is that HO-1-mediated impaired resistance to infection 19 , either linked to or independent of IL-10, could be a significant cause of mortality in sepsis. Further, bacterial infection, per se, may also induce HO-1, as lipopolysaccharide can induce HO-1 via the transcription factor Nrf2 50 .
Understanding the cellular sources of HO-1 and the mechanisms by which it impairs immune function will also be important. Neutrophils are key to the immune defence against invasive bacteria. During sepsis, delayed neutrophil apoptosis 51 accompanied by increased release of neutrophils into circulation 52 results in neutrophilia.  Nevertheless, neutrophil function is markedly diminished due to premature release of immature neutrophils from bone marrow 52 ; these immature cells migrate sub-optimally in response to cytokines and nitric oxide and have reduced capacity to produce reactive oxygen species (impaired oxidative burst) [53][54][55] . This release of defective neutrophils from bone marrow is mediated in large part by the HO-1 pathway 19,56 . Although the underlying   Table 2.
Hazard ratios comparing mortality rates in different subgroups defined by immunological and clinical characteristics at admission. ¶ Unadjusted hazard ratio (HR) comparing individuals above the median to those below the median, ₡ Hazard ratio adjusted for log-transformed IL-10 response.
SCIENTIFIC REPoRtS | (2018) 8:11198 | DOI:10.1038/s41598-018-29558-5 defect in neutrophil maturation has not been characterized in sepsis, we speculate that HO-1 (or the products of HO-1 enzymatic activity) may impact innate immunity. Further, if similar, persistent, HO-1-mediated neutrophil dysfunction occurs in bacterial sepsis, this may begin to explain the increased risk of recurrent infection in sepsis survivors [57][58][59] : up to 63% of sepsis survivors experience at least one subsequent episode of invasive bacterial disease within 12 months and this is fatal in 16.1% of cases 59 . Further work is needed to assess the causal link, if any, between inflammation, IL-10, HO-1 and neutrophil dysfunction in sepsis. Implicating this pathway in the poor prognosis of sepsis patients offers options for improved management of sepsis patients and survivors. In the acute phase of disease, constraining the enzymatic activity of HO-1 by administration of the competitive inhibitor tin protoporphyrin IX (SnPP) 60 may ameliorate the deleterious impacts on neutrophil function whereas in the convalescent phase, neutrophil respiratory burst assays may identify patients at particular risk of relapse or reinfection.
In conclusion, HO-1 is markedly elevated among sepsis patients. Interestingly, although many sepsis patients displayed evidence of haemolysis, IL-10 (as a marker of regulation of inflammation) appeared to be the main driver of HO-1 induction. High levels of HO-1 and IL-10 at admission were predictors of disease severity and mortality. Given the known impact of IL-10 and HO-1 on diminished phagocyte function, the role of HO-1 in sepsis warrants further investigation. Complete blood counts (CBC) were performed using a DxH800 haematology analyser (Beckman Coulter). Plasma samples were aliquoted and stored at −80 °C until use. Identifying patient information was removed prior to sample distribution to the authors.
Plasma protein quantification. Enzyme-linked immunosorbent assays (ELISA) were conducted according to manufacturers' instructions to measure plasma concentrations of haptoglobin (HPT, GWB-8DA44B, Genway Biotech), haemopexin (HPX, GWB-4B6D1A, Genway Biotech), haem oxygenase-1 (HO-1, Figure 5. Proposed model of the relation between haemolysis, IL-10, HO-1 and mortality at admission of sepsis. The majority of sepsis patients at admission are moderately anaemic (low haemoglobin); however, those with the highest erythrocyte counts show elevated markers of haemolysis (haem and haemopexin, HPX) and elevated EPO. Further, systemic infection leads to robust inflammation and correlated with haemolysis (depleted haemopexin levels). Next, we observed that both IL-10 and haemolysis (depleted haemopexin) leads to the induction of haem-oxygenase 1 (HO-1). After partial correlation analysis for HO-1 induction which adjusted for either factor (IL-10 or haem and HPX), we found that IL-10 was the main driver of HO-1. IL-10 and HO-1 both individually showed a significant and moderate correlations to clinical severity score (APACE II, SOFA). Finally, high levels of IL-10, HO-1, and haem (i.e., above median value) at admission were positively associated with in-hospital mortality. Of note, this proposed model is limited to the ICU admission parameters measured in this study -long-term interaction pathways and their full consequences is currently unclear. The causal model (i.e., the direction of the arrows) was determined a priori and tested using the study data, Arrow size: 1 pt (p = 0.05-0.02), 3 pt (p = 0.019-0.001), and 6 pt (p < 0.001).
Statistical analysis. Pearson's correlation was used to identify statistical associations between markers of inflammation (IL-6, TNF-alpha, G-CSF, IL-10), markers of haemolysis (haem, HPX, HO-1) and measures of disease severity (APACHE II). In addition, we calculated partial correlations to adjust for confounding, after having selected potential confounders by applying the "back-door criterion" 62 to the causal diagram in Fig. 5. Interpretation of correlation coefficients (r values) was as described by Ratner 63 ; notably moderate (r = 0.3-0.7) and weak (r < 0.3). Kaplan-Meier curves and hazard ratios were used to investigate associations between mortality and clinical and immunological characteristics at admission. Analyses were conducted using GraphPad Prism 7 and Stata version 14.
Data availability. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.