Olfactomedin 4 Is a Biomarker for the Severity of Infectious Diseases

Abstract

Biomarkers of infectious diseases are essential tools for patient monitoring, diagnostics, and prognostics. Here we review recent advances in our understanding of olfactomedin 4 (OLFM4) in neutrophil biology and of OLFM4 as a new biomarker for certain infectious diseases. OLFM4 is a neutrophil-specific granule protein that is expressed in a subset of human and mouse neutrophils. OLFM4 expression is upregulated in many viral and bacterial infections, as well as in malaria. OLFM4 appears to play an important role in regulating host innate immunity against bacterial infection. Further, higher expression of OLFM4 is associated with severity of disease for dengue virus, respiratory syncytial virus, and malaria infections. In addition, higher expression of OLFM4 or a higher percentage of OLFM4 + neutrophils is associated with poorer outcomes in septic patients. OLFM4 is a promising biomarker and potential therapeutic target in certain infectious diseases.

Infectious diseases are caused by pathogenic microorganisms such as viruses, bacteria, fungi, or parasites [1]. The search for and discovery of new biomarkers for infectious diseases will help clinicians determine patient prognosis, predict clinical outcomes after therapy, and develop new drugs to use in combatting these diseases [2].

Olfactomedin 4 (OLFM4) is a member of a well-conserved olfactomedin domain–containing glycoprotein family [3]. It is endogenously expressed in neutrophils, intestinal crypts, and the prostate and plays important roles in innate immunity, inflammation, and carcinogenesis [4].

OLFM4 is a bona fide target gene of the NF-κB pathway and responds to a variety of microbial infections [4]. Using a Olfm4-deficient mouse model, Olfm4 has been shown to inhibit the NOD1- and NOD2-mediated NF-κB pathway through a feedback mechanism, and removal of Olfm4 reduced Helicobacter pylori colonization in mouse gastric mucosa [4]. Mouse neutrophils without Olfm4 demonstrated increased intracellular bacteria killing due to enhanced cathepsin C and its downstream serine proteinase activity [4].

Its biological characterization, expression, and function in some cancers, especially gastrointestinal and prostate cancers, have been well reviewed previously [4]. OLFM4 is expressed in the intestinal stem cells and is a target gene of the Wnt pathway [4]. OLFM4 competes with Wnt ligand receptors and inhibits the Wnt pathway [4]. Olfm4 deletion induced colon adenocarcinoma in Apc mutant mice and enhanced azoxymethane/dextran sodium sulfate–induced intestinal inflammation [4].

This review focuses on OLFM4’s biology in neutrophils and its upregulation and value as a biomarker in certain infectious diseases.

OLFM4 IN NEUTROPHIL BIOLOGY

OLFM4 was first cloned from human hematopoietic myeloid precursor cells induced by granulocyte colony-stimulating factor [3]. OLFM4 mRNA is expressed during myelopoiesis, with that expression peaking at the immature myelocyte/metamyelocyte stage of differentiation and then declining as the cells mature [5]. In bone marrow and peripheral blood, OLFM4 is exclusively expressed in neutrophils in humans and mice [3, 6].

Olfm4 is not essential for neutrophil development, phagocytosis, and migration, as demonstrated by 2 separately established Olfm4-deficient mouse models [6, 7]. However, a recent study showed that Olfm4 is involved in hydrogen peroxide–induced NADPH oxidase activation and apoptosis in mouse neutrophils [8].

OLFM4 Is a Neutrophil-Specific Granule Protein and Is Located in Neutrophil Extracellular Traps and Exosomes

OLFM4 is a neutrophil-specific granule protein [5]. Under stimulation, it is associated with neutrophil extracellular traps (NETs) [6, 9]. OLFM4 is located within the webs of DNA in neutrophils of humans and mice [6, 9]. The functional role of OLFM4 in NETs is still unknown, although it has been hypothesized that OLFM4 may be involved in NETosis [9].

A proteomic profiling study in human neutrophil exosomes found that neutrophils can secrete OLFM4 via exosomes [10]. OLFM4 was identified as the critical differentially expressed protein in generalized pustular psoriasis (GPP) and was much more abundant in neutrophil exosomes from GPP than from control neutrophil exosomes. A higher protein level of OLFM4 was detected in the sera of patients with GPP than in controls; higher serum protein levels of OLFM4 also positively correlated with GPP severity score and decreased after effective treatment in patients. This study demonstrates that OLFM4 mediates the autoimmune inflammatory response in GPP [10].

OLFM4 Is Expressed in a Subset of Human and Mouse Neutrophils

OLFM4 is expressed in a subset of neutrophils [5, 9]. In healthy human volunteers, 1 study reported that 20%–25% of neutrophils from peripheral blood express OLFM4 [5], while another study demonstrated a wider variance in OLFM4 + neutrophil populations (8%–57%) [9]. No difference in phagocytosis, migration, or apoptosis between OLFM4 + and OLFM4 − neutrophil populations from humans has been identified [9].

Depending on the strain, 7%–35% of mouse neutrophils have been found to express OLFM4, and this expression is determined early in neutrophil differentiation [6]. As in human neutrophils, OLFM4 + and OLFM4 − mouse neutrophils have been reported to phagocytose and transmigrate with similar efficiency [6].

Despite this documented heterogeneity of OLFM4 expression within neutrophils, it is difficult to distinguish whether these different OLFM4 expression subpopulations represent altered activation states of a single population or separate subpopulations of neutrophils determined at the time of differentiation. One limit for studying the differential functions of such OLFM4 expression–defined subsets of neutrophils is that neutrophils are killed after the fixation and permeabilization methods that are used to stain intracellular OLFM4.

OLFM4 IS A BIOMARKER FOR THE SEVERITY OF VIRAL INFECTION

Effective antiviral drugs for many viral infections have not yet been discovered. Therefore, identification of predictive biomarkers for viral infections is needed to improve disease intervention and management. OLFM4 is emerging as a biomarker for the severity of several viral infections.

Dengue Virus

Patients infected with dengue virus usually present a mild, self-limiting febrile dengue infection (DI) that occasionally leads to a potentially lethal complication, called severe dengue (DS). To identify prognostic markers for DS, RNA isolated from the peripheral blood mononuclear cells of dengue patients with varying levels of severity of disease, patients with other febrile illnesses, or healthy controls was subjected to high-throughput sequencing [11]. A comparative analysis determined that OLFM4 and other neutrophil activation proteins (such as MPO and ELANE) were upregulated in DS, but not DI [11]. These results suggest that OLFM4 may be valuable for the prognosis and management of dengue patients [11].

In another study to search for predictive markers for DS, the proteomes of virion-enriched fractions purified from plasma pools of patients with severe dengue fever or dengue fever were compared using nano-liquid chromatography coupled to ion trap mass spectrometry [12]. Among 22 host proteins tested, OLFM4 and platelet factor–4 could differentiate between severe dengue fever and dengue fever in 2 independent cohorts [12]. The protein concentration of OLFM4 was higher for severe dengue patients than dengue fever patients [12]. Therefore, measuring patient serum OLFM4 concentration will provide prognostic value to identify patients with dengue who are likely to develop severe dengue [12].

Respiratory Syncytial Virus

Respiratory viral infections follow an unpredictable clinical course in young children, ranging from a common cold to respiratory failure. Thus, an urgent need exists to discover biomarkers that can predict the transition from mild to severe disease.

Genome-wide gene expression analyses were performed on peripheral blood mononuclear cells of 26 children with a diagnosis of severe, moderate, or mild respiratory syncytial virus (RSV) infection [13]. OLFM4 was the most upregulated gene and was upregulated >40-fold in severe infection [13]. In addition, patients with a higher OLFM4 gene expression level were 6 times more likely to develop severe disease after correction for age at hospitalization and gestational age [13]. This study identified OLFM4 as a fully discriminative marker between children with mild and severe RSV infection [13].

HIV

Tuberculosis (TB) is the most common cause of AIDS-related deaths. A series of bioinformatics analytical methods was used to identify genes involved in the mechanisms of HIV and Mycobacterium tuberculosis coinfection [14]. Among a total of 113 HIV/TB and 109 HIV/latent TB infection genes selected, OLFM4 was 1 of the 12 significantly upregulated genes in HIV/TB patients [14].

Vaginal microbicides have been used to prevent the transmission of viral diseases like HIV. To identify biomarkers that can accurately predict microbicide toxicity early in preclinical development, quantitative proteomics and reverse transcription polymerase chain reaction approaches have been used to identify changes in vaginal fluid and tissue in response to treatment with nonoxynol-9 (N9) or benzalkonium chloride (BZK) in mice and rabbits [15]. OLFM4 was downregulated in vaginal tissue by N9 or BZK treatment [15]. Furthermore, using a human vaginal epithelial cell line, the expression of OLFM4 was downregulated in response to N9, suggesting that this marker could be relevant in humans [15]. These data indicate that OLFM4 could be useful as a biomarker to evaluate microbicide toxicity in vaginal HIV infection [15].

OLFM4 RESPONSE AND ROLES IN BACTERIAL INFECTION

Bacterial infections represent a significant cause of morbidity and mortality in the world, especially among children. Exploring the gene transcription signature will contribute to understanding infection pathogenesis and be of benefit to clinical therapy. OLFM4 is upregulated in several gram-positive and gram-negative bacterial infections and plays an important role in the host’s innate immunity against bacterial infection.

Helicobacter Pylori

OLFM4 expression was found to be upregulated in the gastric antral mucosa of H. pylori–infected patients [16]. The role of Olfm4 in H. pylori gastric mucosa infection has been studied in an Olfm4-deficient mouse model [7]. H. pylori colonization in the gastric mucosa of Olfm4-deficient mice was significantly lower than that observed in wild-type (WT) littermates [7]. Furthermore, enhanced immune response and inflammation were observed in Olfm4-deficient mice upon H. pylori infection [7]. This study also showed that Olfm4 inhibits NOD1- and NOD2-mediated NF-κB activation, suggesting that Olfm4 plays an important role in regulating innate immune responses [7].

Escherichia coli and Staphylococcus aureus

Increases in E. coli and S. aureus bloodstream infections and antimicrobial resistance are still challenging hospitals worldwide. An analysis of the distinct gene expression signature induced by bacterial and viral infections in peripheral blood leukocytes revealed a statistically significant upregulation of OLFM4 in children, aged 0–16 years, diagnosed with bacterial infections (including E. coli and S. aureus) or influenza A virus compared with healthy controls [17]. In this cohort, children with S. aureus infections had statistically significantly higher OLFM4 gene expression compared with influenza A–infected patients [17]. In human colon epithelial LS174T cells, OLFM4 mRNA was considerably upregulated after incubation with E. coli K12, E. coli Nissle, or Bacteroides vulgatus [18]. Neutrophils from Olfm4-deficient mice have been reported to have increased capability to kill S. aureus and E. coli and are more resistant to systemic sepsis than neutrophils from WT mice [19]. The mechanism of increased killing of bacteria in Olfm4-deficient neutrophils has been attributed to Olfm4 binding and inhibition of cathepsin C, a serine protease that potentiates several cellular antimicrobial functions [19]. However, other mechanisms may also play a role, because crossing the Olfm4-deficient mouse onto cathepsin C–deficient mice still protected from challenges by E. coli and S. aureus in 1 study, and only 6%–8% of neutrophils express Olfm4 in C57BL6 mice [6].

The impact of Olfm4 deletion on host defense against S. aureus has been examined in a mouse X-linked gp91phox deficiency chronic granulomatous disease (CGD) model [20]. Intracellular killing and in vivo clearance of S. aureus, as well as resistance to S. aureus sepsis, were significantly increased in gp91phox Olfm4 double-deficient mice compared with CGD mice [20]. These results showed that Olfm4 deletion could enhance immune defense against S. aureus in CGD mice, suggesting that OLFM4 may represent a potential target to boost host defense against bacterial infection in CGD patients [20].

Lawsonia intracellularis

L. intracellularis is a gram-negative, obligate intracellular bacterial pathogen that causes proliferative enteropathy in pigs. To understand the mechanism of this disease, gene expression profiles of L. intracellularis inction were determined, and Olfm4 was found to be upregulated in the intestinal crypts [21]. The Notch and Wnt/β-catenin signaling pathways were also found to be activated by L. intracellularis induction [21]. The significant upregulation of the Olfm4 transcript, which encodes for a robust intestinal stem cell marker associated with anti-apoptotic effects, supports the notion that genetic reprogramming of intestinal stem cells occurs to enhance their survival during the early stages of crypt regeneration after damage by L. intracellularis infection [21].

Porphyromonas gingivalis

Oral epithelial cells are central to maintaining homeostasis in the face of a diverse and abundant microbiota. High-throughput sequencing analysis of RNA isolated from human oral epithelial cells showed that the Notch signaling pathway, including the downstream effector OLFM4, was upregulated by the oral pathogen P. gingivalis [22]. A concomitant increase in OLFM4 protein levels in response to P. gingivalis was also observed in these cells. This investigation further found that the Notch1-Jag1 signaling cascade was the major pathway through which OLFM4 was regulated by P. gingivalis [22]. OLFM4 was determined to be required for epithelial cell migratory, proliferative, and inflammatory responses to P. gingivalis. Thus, OLFM4 is an important player in the oral bacteria–host interaction [22].

OLFM4 IS A PROGNOSTIC FACTOR FOR SEPSIS AND POSTSURGICAL SHOCK

Growing evidence indicates that neutrophil-related pathways may be directly linked to the development of sepsis-related organ failure and death [23]. So far, no reliable prognostic system exists to predict the death risks in patients with sepsis or postsurgical shock. However, several recent clinical studies have found that higher expression of OLFM4 in blood samples and a higher percentage of OLFM4 + neutrophils are associated with worse outcomes in patients with sepsis and postsurgical shock. In addition, OLFM4 gene polymorphisms have recently been identified as potential mediators of postsurgical septic shock mortality [24].

Higher Expression of OLFM4 Is Associated With Worse Outcomes in Patients With Sepsis and Postsurgical Shock

A study of the expression of OLFM4 in children with septic shock found that OLFM4 is the most highly differentially expressed gene in nonsurvivors compared with survivors [25]. OLFM4 serum protein concentrations in pediatric patients with septic shock were also found to be higher than those in healthy pediatric controls [25]. In addition, the median OLFM4 serum concentration was observed to be significantly higher in the septic shock patients with a challenging course than in those patients without a complicated course [25]. This study demonstrated that OLFM4 correlated with the severity of septic shock at both the mRNA and protein levels [25].

In another clinical study, gene expression signatures were examined to distinguish between low and high risk of death in postsurgical shock patients [26]. mRNA microarrays of whole-blood samples were analyzed to identify genes that were differentially expressed between the surviving and nonsurviving groups 30 days after the operation. OLFM4, CD177, RETN, and IL1R2 were identified as the top 4 upregulated genes in the nonsurviving group [26]. This gene expression cluster can predict mortality better than classical mortality risk scores, such as the Acute Physiology And Chronic Health Evaluation (APACHE) and Sequential Organ Failure Assessment (SOFA) [26]. Thus, OLFM4 represents an efficient biomarker to distinguish between low and high risk of death in patients with postsurgical shock [26].

Distinguishing septic shock and nonseptic shock in postoperative patients is an important goal, because it would eliminate unnecessary use of broad-spectrum antibiotics and the development of antimicrobial resistance. A recent mRNA microarray analysis using whole-blood samples found that OLFM4, as well as IGHG1, IL1R2, LCN2, LTF, and MMP8, were upregulated in septic shock postsurgical patients compared with non–septic shock postsurgical patients [27]. Furthermore, this gene expression pattern was shown to be a better biomarker than procalcitonin and C-reactive protein, the 2 biomarkers most used for the diagnosis and evolution of septic shock [27]. Thus, OLFM4 shows potential to be a valuable biomarker to aid in the immediate and specific diagnosis and treatment of septic shock [27].

Acute kidney injury is a common complication of sepsis, and its occurrence is a poor prognostic sign in septic patients. Bioinformatics analysis has identified OLFM4 as significantly associated with the occurrence and development of acute kidney injury in septic shock patients and suggests that OLFM4 could become a potential novel marker for septic patients with kidney damage [28].

Another common and early complication of sepsis is acute respiratory distress syndrome (ARDS). To understand the mechanism of development of ARDS in patients with sepsis, gene expression was examined in whole-blood samples from patients with sepsis and ARDS and patients with sepsis alone [29]. Analysis of gene expression profiles identified OLFM4 as the most highly differentially expressed gene in septic patients with ARDS compared with patients with sepsis alone, suggesting OLFM4’s potential involvement in the early pathogenesis of sepsis-related ARDS [29].

A Higher Percentage of OLFM4-Positive Neutrophils Predicts Worse Survival in Sepsis Patients

In a study of patients with septic shock in which the percentage of OLFM4 + neutrophils was quantified, patients with a complicated course (defined as ≥2 organ failures at day 7 of septic shock or 28-day mortality) had a higher percentage of OLFM4 + neutrophils than septic shock patients without a complicated course [25]. Furthermore, logistic regression analysis indicated that the percentage of OLFM4 + neutrophils was independently associated with an increased risk of organ failure and death [25]. Therefore, OLFM4 may be useful as a marker of a pathogenic neutrophil subset in patients with septic shock [25].

In a single-center, prospective cohort study, 56% of sepsis patients with a higher percentage of OLFM4 + neutrophils died, compared with 18% of sepsis patients with a lower percentage of OLFM4 + neutrophils [30]. Further, this study found that OLFM4 + neutrophil percentage was independently associated with 60-day mortality in sepsis patients and may therefore represent a novel measure of the heterogeneity of host response to sepsis [30]. In addition, a higher percentage of OLFM4 + neutrophils was associated with shock (72% vs 47%) and respiratory failure requiring mechanical ventilation (67% vs 38%) [30]. These data indicate that the OLFM4 + neutrophil subset can be used as a measure of a biological response that is distinct from clinical measures of neutrophil numbers and maturity, as well as several plasma measures of inflammation and injury, in human sepsis [30].

These clinical studies established the association between the percentage of OLFM4 + neutrophils and the death risk in patients with sepsis. However, it is not clear whether the OLFM4 + percentage increases in response to the severity of illness in sepsis or if OLFM4 + neutrophils are pathogenic in response to sepsis. Nevertheless, the percentage of OLFM4 + neutrophils may serve as an important pathogenic biomarker that could be useful for the identification of specific clinical treatments.

Olfm4-Deficient Mice Are Protected From Sepsis

The role of Olfm4 in sepsis has also been studied in mouse models [6, 19, 31]. Two separate studies have demonstrated that Olfm4-deficient mice were protected from death when challenged with sepsis induced by cecal ligation and puncture model [6] or with intraperitoneal injection of lethal doses of E. coli and S. aureus [19]. These studies together suggest that Olfm4 may play a pathogenic role in the host immune responses to sepsis.

In another study in which sepsis was induced in murine pups, survival was significantly increased in Olfm4-deficient pups compared with WT pups [31]. After induction of sepsis, increased expression of Olfm4 was observed in the kidneys of WT pups compared with those of placebo-treated WT pups and was localized to the loop of Henle. In addition, renal cell apoptosis and plasma creatinine were significantly increased in WT pups when compared with Olfm4-deficient pups [31]. Bone marrow transplant suggested that the increased Olfm4 expression observed in the kidneys of WT pups reflects local production of Olfm4 rather than Olfm4 filtered from plasma [31]. These results demonstrate renal expression of Olfm4 for the first time and suggest that a kidney-specific mechanism may contribute to the survival differences observed in Olfm4-deficient mice compared with WT mice [31].

OLFM4 GENE EXPRESSION IS ASSOCIATED WITH SEVERE MALARIA

Plasmodium falciparum malaria is one of the most important infectious diseases affecting humans [32]. To gain insight into host–pathogen interactions associated with severity of malaria infection in humans, a dual RNA sequencing analysis of human and malaria differential gene expression in whole blood from patients with either severe or uncomplicated P. falciparum malaria was performed [33]. OLFM4 and other neutrophil granule proteins, including MMP8, DEFA3, and ELANE, were found to be highly expressed in severe malaria patients when compared with uncomplicated malaria patients. These results support a putative role for neutrophils in severe malaria [33]. Because the release of neutrophil granule proteins can be highly damaging to host tissues [34], increased production and release of these proteins may contribute to many of the pathological features of severe malaria. The observed association of neutrophil granule protein genes with severe malaria suggests that targeting neutrophil function may be a therapeutic strategy for malaria in the future.

CONCLUSIONS

As a neutrophil granule protein, OLFM4 is emerging as a promising biomarker to predict the clinical outcome of patients with certain infectious diseases. However, the molecular mechanism for OLFM4’s involvement in this process remains to be further investigated. In addition, exploring effective inhibitors of OLFM4 may prove beneficial therapeutically for those infectious disease patients with a high level of OLFM4.