I-FABP is a member of the FABP family, has a molecular weight of approximately 15 kilodaltons (kDa), and plays a key role in the transportation and metabolism of long-chain fatty acids. It is specific to and abundant in the epithelial cells of the small intestine (102). Upon intestinal mucosal injury, I-FABP is released from epithelial cells into the circulating blood, and levels of plasma I-FABP have been reported to correlate with the severity of intestinal disease [e.g., ulcerative colitis, Crohn’s disease (CD), mesenteric ischemia, coeliac disease], and is considered a marker of intestinal epithelial cell tight junction disruption and cell death (103–107).

I-FABP has been shown to be an indicator of intestinal damage in PLWH. One prospective trial of ART-naïve HIV-infected individuals observed a remarkable elevation in I-FABP from baseline to week 96, and this elevation correlates with viral replication (108). Sustained effective ART significantly reduces immune activation and tends to ameliorate peripheral T-cell immunophenotypic imbalances; however, this positive and beneficial immunological response to targeted drug therapy is observed to be insufficient to adequately and comprehensively restore intestinal permeability and integrity, and plasma I-FABP levels remain markedly elevated subsequent to ART treatment (109). In chronic HIV-infected patients, I-FABP is increased to a much greater degree than in elite controllers and correlates with nutritional intake and body composition (body mass index, visceral and subcutaneous adipose tissue distribution) in HIV progressors (90). As an intestinal epithelial barrier marker, I-FABP may also act as a predictor of mortality in treated HIV-infected patients. Hunt et al., reported that, similar to other markers of intestinal barrier integrity (Zonulin-1) and some inflammatory factors, I-FABP strongly predicts mortality in treated HIV-infected individuals, with higher plasma I-FABP levels being associated with higher soluble CD14 (sCD14) levels, kynurenine-to-tryptophan (K/T) ratios, IL-6, and D-dimer levels, and lower proximal CD4⁺ T-cell counts (110). It is also worth noting that levels of plasma I-FABP have also been reported to correlate with other non-inflammatory factors (111–113). Our previous study observed that levels of I-FABP were affected by circadian rhythm, and plasma levels of I-FABP at 16h00 were significantly lower, compared to levels at 08h00 and 04h00 (111). Auinger et al., have demonstrated that I-FABP expression significantly correlates with fatty acid intake in food as well as I-FABP gene polymorphisms (112).

Zonulin is a protein with a molecular mass of 47 kDa, is specifically expressed at the cell surface and secreted by the intestinal epithelium, and is the only physiological enzyme discovered thus far that is known to regulate intestinal permeability via reversibly disassembling the intercellular tight junctions between intestinal epithelial cells (114–116). Serum zonulin has been reported to be elevated in inflammatory and autoimmune disorders [e.g., type 1 diabetes, inflammatory bowel disease, ankylosing spondylitis (AS), and CD], and its levels correlate with the opening of tight junctions and increased intestinal permeability when zonulin production is dysregulated (117–119). It has also been observed that the synthetic TJ regulator larazotide (which acts in an inhibitory capacity) blocks zonulin enzyme activity with excellent efficacy in type 1 diabetes and celiac disease (118, 120, 121).

The zonulin-driven intestinal cellular immune response has been validated during in vitro experiments (122). When the small intestine is exposed to microbes, there is an increase in the secretion of zonulin from the intestinal lumen. Zonulin disassembles TJs by separating the zonula occludens-1 protein from the TJ complexes, decreasing transepithelial electrical resistance (TEER), and increasing permeability (122). Likewise, plasma zonulin levels are elevated in PLWH and are associated with monocyte and T-cell activation (92, 123). Perinatally HIV-infected infants show increased circulating levels of zonulin despite viral suppression by treatment with early ART, and these levels are significantly higher than in HIV-exposed but uninfected infants at 5 months of age (124). Compared to chronically HIV-infected patients, plasma zonulin levels in primary HIV infection (PHI) are significantly elevated and correlate with high viral replication. Plasma zonulin also demonstrates the best accuracy to identify PHI among HIV-infected individuals {AUC=0.85 [95% CI 0.75-0.94]}. Using a cutoff value of plasma zonulin >8.75 ng/mL, the model identified PHI with 87.7% sensitivity and 69.2% specificity (93).

Additionally, elevated zonulin levels have also been associated with disease progression in PLWH. Hunt et al., reported that zonulin has the strong capacity to predict mortality in treated PLWH who had an AIDS history (110). In perinatally HIV-infected children with a history of breastfeeding, zonulin is associated with multiple markers of systemic inflammation, including CRP, IL-6, and D-dimer (92). Improvement and amelioration of the intestinal barrier via serum bovine immunoglobulin significantly reduces circulating levels of I-FABP and zonulin, and systemic inflammation is also ameliorated (125).

REG3α, is a C-type lectin antimicrobial peptide secreted by Paneth cells in the gut lumen, and helps contain bacterial infection by binding to peptidoglycans in the cell wall of certain bacteria, and has the capacity to kill some gram-positive bacteria (126, 127). REG3α also helps maintain intestinal barrier integrity by reducing apoptosis of intestinal epithelial cells (128). When the integrity of the intestinal epithelial barrier is disrupted, REG3α crosses through the epithelium, translocates to the lamina propria, and subsequently enters into the systemic circulation (28, 129). Thus, circulating REG3α levels are considered to be a marker of intestinal permeability.

REG3α has long been proposed as a biomarker of intestinal epithelial damage in multiple inflammatory diseases in which plasma REG3α levels are significantly elevated, such as CD, celiac disease, ulcerative colitis, nonalcoholic steatohepatitis, and gastrointestinal graft-versus-host disease (129–133). Heavy alcohol consumption is known to disrupt the integrity of the gut epithelium. Yang et al., used REG3α and Trefoil factor 3 as biomarkers to assess intestinal damage and microbial translocation in patients with alcoholic hepatitis, and observed that circulating levels of these markers are highly elevated, and differentially correlates with disease severity, sCD14 levels, and IL-6 levels (134). Darnaud et al., studied the mechanism by which REG3α maintains intestinal homeostasis and affects inflammatory responses in genetically engineered C57BL/6 mice. The preceding authors observed that REG3α is a potent reactive oxygen species (ROS) scavenger that reduces oxidative stress and inflammatory responses in intestinal epithelial cells, reduces host susceptibility to colitis, and alters the murine gut microflora composition (inducing an increase in Clostridium and a decrease in Bacteroides and Proteus) by reducing ROS levels (135).

Given that REG3α is a reliable marker of intestinal barrier damage, REG3α has been also used in HIV patients to assess the degree of gut damage and systemic immune activation. As reported by Isnard et al., plasma REG3α levels are elevated in untreated and ART-treated PLWH (including elite controllers), when compared to HIV-negative individuals. In contrast, plasma REG3α levels are decreased in PLWH who initiate ART, compared with untreated patients. REG3α levels also negatively correlate with CD4⁺ T-cell counts and CD4⁺/CD8⁺ ratios, and positively correlate with HIV viral load, fungal translocation products, and inflammatory markers in all PLWH (28). Immune non-responders (INRs) are PLWH who fail to adequately restore CD4⁺ T-cell numbers after effective ART and are known to have irreversibly impaired intestinal mucosal barrier function. It has been observed that INRs have higher levels of plasma I-FABP and REG3α, compared to immune responders (136), and that mucosal CD4⁺ T-cells positively correlate with I-FABP and REG3α (91).

Citrulline is a non-essential amino acid synthesized from glutamine, and plays a role associated with inflammatory disease (137–142). In several organ exclusion experiments, it has been observed that most citrulline is synthesized in the intestinal epithelium, and subsequently enters into the blood circulation; thus, the intestinal epithelium is the main source of circulating citrulline under physiological conditions (138, 143–145). When intestinal epithelial injury occurs, the production of citrulline decreases, and consequently, in contrast to the trend seen for other biomarkers, the plasma concentration of citrulline decreases.

Circulating citrulline has been used to estimate the degree of intestinal injury, and circulating levels negatively correlate with disease severity in intestinal enteropathies (94, 137, 146, 147). Crenn et al., analyzed the relationship between plasma citrulline concentrations and villous atrophy in celiac disease (148). Their results showed that plasma citrulline concentration is lower in patients with villous atrophy than in healthy subjects, and negatively correlates with the severity of villous atrophy. The results of an investigation in pediatric and adolescent cases of CD by Diamanti et al., showed that CD patients have a reduced concentration of plasma citrulline compared to controls, and that plasma citrulline levels are significantly lower in patients with small bowel localization of CD than in patients with ileo-colon disease (149). Nuzzo et al., analyzed plasma citrulline concentrations in acute mesenteric ischemia (AMI), and plasma citrulline concentrations were observed to be significantly lower in AMI patients compared to controls; however, its practicality for the diagnosis of AMI was found to not be satisfactory, with an area under the receiver operating curve (AUROC) sensitivity and specificity of 0.68, 56%, and 84%, respectively (137). Kulu et al., also observed similar results, with plasma citrulline concentrations in AMI patients being lower, and the AUROC sensitivity and specificity for the diagnosis of AMI were 0.72, 39%, and 100%, respectively, with the best cut-off value being 15.82 µmol/L (150). Furthermore, Fragkos et al., used a meta-analytical method to assess citrulline as a biomarker of gut damage (147), and their results observed that plasma citrulline negatively correlates with disease severity in intestinal enteropathies. The preceding authors have advocated for the use of citrulline as a marker for acute and chronic intestinal insufficiency. In PLWH, Papadia et al., reported that median citrulline levels in HIV positive individuals is significantly lower than that in HIV-negative individuals, and that there are statistically significant correlations between citrulline and villous atrophy in HIV positive individuals (94). Thus, citrulline has been identified as a biomarker for intestinal injury; however, further studies are warranted in order to validate its accuracy among different subgroups of PLWH.

Human diamine oxidase (hDAO) is encoded by the amine oxidase copper containing 1 (AOC1) gene, located on chromosome 7q35 (151). DAO is mainly expressed in the human intestinal mucosal epithelium, the placenta, and the kidney, and catalyzes the oxidation of diamines such as histamine, putrescine, and cadaverine. High activity of this enzyme is found in the mature upper villus cells of the intestinal mucosa, and the intestine is the sole source of plasma diamine oxidase (152–156). DAO is normally present in exceedingly small quantities in the systemic circulation, is stable in the circulation, and high plasma levels are associated with poor integrity of the intestinal mucosa (156, 157).

DAO has been used as a serum marker of intestinal injury (156, 157). It has been reported that plasma DAO activity is associated with the histological and biochemical changes indicative of injury and recovery in a rat model of intestinal injury (155). DAO activity has also been observed in some diseases causing intestinal impairment in humans, including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) (158–161). Meng et al., studied the intestinal injury induced by high dose methotrexate in children with acute lymphoblastic leukemia (158), and they observed that levels of plasma LPS and DAO at 1h, 24h, and 44h gradually increased after treatment (158). Zhang et al., reported a similar tendency in DAO levels in patients with heatstroke (160). Additionally, Ji and colleagues observed higher DAO activity levels in 60 patients with IBS compared to 20 healthy controls, and DAO activity levels correlated with disease severity (162). It is worth noting, however, that some investigations observed decreased plasma DAO levels in some severe instances of intestinal disease, such as IBD (161, 163), and anticancer drug treatment (95, 164). A probable reason that may explain these contrasting observations could be related to the fact that DAO-producing intestinal epithelial cells were significantly reduced in the latter specific instances (163, 165, 166). Moreover, several factors may affect DAO activity, such as genetic variability, the pH-value and temperature of the surrounding milieu, and medication use (167–169). Ayuso et al., identified three variants (Thr16Met, Ser332Phe, and His645Asp) of the AOC1 gene, and reported that individuals carrying the His645Asp variant displayed lower serum DAO activity as compared with noncarriers, with a significant gene-dose effect (168). In vitro experimental results have observed a potent DAO inhibitory effect (of greater than 90%) associated with chloroquine and clavulanic acid (167).

It is, therefore, suggested that DAO may be used as a candidate biomarker to evaluate the extent of intestinal injury when few other factors may be discernable, especially in the initial stages of intestinal injury.

Lipopolysaccharide (LPS), a critical component of the cell wall of gram-negative bacteria, consists of hydrophobic lipids and hydrophilic sugars, and is soluble in both water and lipids, thus facilitating diffusion (170, 171). The intestinal barrier prevents microorganisms and their products, such as LPS, from entering into the systemic circulation (36, 48). In PLWH, however, the disrupted intestinal barrier together with an increased permeability allows LPS to translocate into the blood circulation (36). In addition, when the monocyte/macrophage receptor for LPS, i.e., CD14, binds with circulating LPS, this induces activation and systemic inflammation (172, 173). CD14 is cleaved and released upon cell activation. Plasma LPS and sCD14 levels are increased in PLWH (36), and plasma levels of sCD14 have been generally shown to positively correlate with circulating LPS levels (174). Thus, circulating LPS and sCD14 have been frequently utilized as biomarkers of bacterial translocation, and have been observed to be associated with systemic immune activation and HIV disease progression (34, 171, 173, 175, 176). Nevertheless, some studies have also reported that LPS either negatively correlates with or does not correlate with sCD14 at all (177–179). This ambiguity may relate either to the CD14 genomic polymorphism (180), multifarious LPSs from different bacteria (181), a different extent of influence from ART, or opportunistic coinfections in these patients (177).

Immunohistochemistry results in SIV-infected rhesus macaques illustrate multifocal compromises and epithelial breaches in the normally intact epithelial barrier in gut tissues. In concert with this, quantitative image analysis of microbial translocation has revealed the emergence of LPS in the gut as well as parenteral tissue of rhesus macaques (1). Numerous studies have shown that levels of LPS and sCD14 are raised in PLWH (2, 34, 96, 175). Brenchley et al., reported that LPS levels are mildly increased in acute/early HIV-infected individuals, compared to HIV-uninfected individuals (2). However, significantly higher LPS levels are observed in chronic HIV-infected individuals, and sCD14 levels are raised in all cohorts of PLWH (2). LPS levels have also been associated with increased activation of activated CD4⁺ and CD8⁺ T-cells and plasma Interferon-alpha (IFN-α) levels (2). Even elite controllers have higher LPS levels than HIV-negative individuals, and the higher plasma LPS levels are associated with higher activated CD8⁺ T-cell counts (97). Furthermore, research by Marchetti et al., observed that compared with immunological responders, immunological non-responders, additionally, had higher LPS levels, which correlates significantly with frequencies of activated CD4⁺ and CD8⁺ T-cells (96).

A robust association has also been observed between clinical outcomes and levels of sCD14 and LPS (182–184). A study by Marchetti et al., observed that in a cohort of 379 HIV-infected individuals, circulating LPS level is a strong predictor of disease progression, independently of CD4⁺ T-cell counts and plasma viral load counts (175). PLWH with higher LPS levels showed a substantially accelerated disease progression rate, with a median time to clinical event of 1.5 years, compared with 4 years for patients with lower LPS levels (175). Jumare and colleagues observed that compared with uninfected controls, plasma levels of sCD14 were significantly higher in PLWH, and among PLWH, those with neurocognitive impairment had significantly higher sCD14 levels compared with neurocognitively unimpaired individuals (182).

Presence of circulating fragments of microbial DNA is generally accepted to be valid evidence for bacterial translocation (185, 186). DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) corresponds to rRNA on bacterial chromosomes, with a length of about 1542 base pairs (bp) (187). 16S rDNA exists in all bacterial chromosomal genes, with homologous functions, and the oldest of these genes (known as “bacterial fossils”) are highly conserved in structure and function (188). As an important bacterial product, 16S rDNA of gut origin is highly likely to translocate into the systemic circulation in the presence of compromised gut integrity (189). Thus, measurement of 16S rDNA levels in plasma has the capacity to effectively reflect levels of microbial translocation (186, 189, 190).

By quantitative PCR (qPCR) determination of 16S rDNA fragments, Jiang et al., observed that plasma 16S rDNA levels in HIV-infected individuals are significantly higher than in uninfected individuals, and correlates with LPS levels (21, 178, 190). Plasma 16S rDNA level increases with duration of HIV infection (98), and treatment with ART may reduce, but does not fully eliminate plasma levels of bacterial 16S rDNA (109). Higher levels of 16S rDNA during treatment are strongly associated with higher T-cell activation and lower CD4⁺ T-cell recovery, regardless of plasma HIV RNA viral load (21). In the context of non-human primate lentiviral infection, 16S rDNA levels and CD8⁺ T-cell activation inversely correlate with the Th17/regulatory T-cell (Th17/Treg) ratio, suggesting that the extent of microbial translocation and T-cell activation in progressive HIV disease is closely related to skewed maturation along the TH17/Treg axis (191). These findings highlight the importance of microbial translocation markers such as LPS and sCD14 in the context of immunodeficiency and T-cell homeostasis in chronic HIV infection (2, 21, 192).

Plasma 16S rDNA is strongly associated with persistent immune activation in HIV disease. Among HIV-Hepatitis C virus (HCV) co-infected patients, bacterial 16S rDNA levels are notably higher than in the ART-controlled HIV-positive group, and 16S rDNA levels have been seen to increase with duration of HIV infection (98, 193). In HIV-positive patients with neuro-inflammation, Jaime et al., observed a significant correlation between plasma concentrations of 16S rDNA and increased expression of translocated proteins in brain regions with markedly active microglia, such as the basal ganglia and the globus pallidus (194). Plasma 16S rDNA concentrations are also associated with increased white matter tract density (194). One study by Bossola et al. (195), observed that plasma levels of bacteria-derived 16S rDNA in whole blood are statistically significantly associated with higher levels of CRP and IL-6 in patients undergoing chronic hemodialysis. Furthermore, 16S rDNA may bind to Toll-like receptors (TLR) and stimulate immune cells (196). These induce Natural Killer (NK) cell activity and the release of IFN-γ, tumor necrosis factor alpha (TNF-α), and IL-6 from mononuclear cells (197–200). Similarly, in treated HIV-infected patients, higher levels of inflammatory markers (IL-6 and TNF-α) are associated with microbial translocation (16S rDNA, sCD14) and previous cardiovascular events (201).

Fungi are also an integral component of the gut microbiome and is second only to bacterial frequency in terms of numbers of organisms (80, 202, 203). Similar to translocation of bacterial products, higher circulating levels of fungal products have also been found in PLWH (44, 123, 204). As a major component of most fungal cell walls, (1, 3) β-D-Glucan (BDG) has now been validated as a fungal translocation biomarker (27, 29), and is contemporarily used for the clinical diagnosis of invasive fungal infections (IFI) (205, 206).

Leelahavanichkul et al., tested serum BDG levels in several murine models of gastrointestinal leakage (including dextran sulfate solution administration, LPS injection, and cecal ligation and puncture sepsis), and observed increased levels of the fungal product, BDG, in the systemic circulation (207). Morris et al., first reported that the fungal product, BDG, is present in the blood of PLWH, and that those individuals with higher plasma BDG are associated with lower CD4⁺ T-cell counts, a higher viral load, and cardiopulmonary comorbidity (208). Subsequently, other investigators also reported higher BDG levels in PLWH (92, 209–211). An investigation by Mehraj et al., showed that, in a similar manner to LPS levels indicating translocation of bacterial products, plasma BDG levels are elevated in all HIV-positive patients without IFI, compared with HIV-negative controls (44). The preceding group prospectively assessed the levels of BDG in early and chronic HIV infection and observed increasing levels over time, in absence of treatment (44). Similarly, a cross-sectional study conducted in Ugandan children showed that compared with HIV-exposed uninfected children, those who acquired HIV perinatally had higher plasma BDG levels that correlated with IL-6 and D-dimer levels (92). A longitudinal investigation in 451 participants who were ART-naïve at baseline showed that BDG levels had decreased after 48 weeks of ART treatment, and higher BDG levels are associated with increased risk of non-AIDS events (209). Hoenigl et al., reported that plasma BDG concentrations are not affected by plant BDG-rich food, further indicating that translocated BDG may be deemed as a reliable marker of intestinal fungal translocation (212). These studies indicate BDG may function as a potential biomarker to indicate whether to initiate antifungal prophylaxis in the early stages of HIV infection, or not.

Higher blood BDG levels have also been reported to correlate with immune activation and risk of developing non-AIDS comorbidities (27, 29, 44, 213). Among PLWH on ART, higher plasma BDG levels are associated with higher levels of activated CD4⁺ and CD8⁺ T-cells, and positively correlates with the K/T ratio (44), which is linked to gut damage and bacterial translocation during HIV infection (214–216). Weiner et al., reported that higher BDG levels are associated with the inflammatory cytokines sCD14, IP-10, D-dimer, and sCD163 (213). A study by Hoenigl et al., reported that higher plasma BDG levels were related significantly to worse neurocognitive performance among HIV-infected individuals with suppressed viral loads (217). An investigation by Isnard et al., observed the association between elevated plasma BDG levels and subclinical coronary atherosclerotic plaques in PLWH. Interestingly, BDG levels (and not LPS levels) were elevated significantly in ART-treated PLWH with subclinical coronary atherosclerosis and correlates with total plaque volume (89).

Flagellin is a structural protein present on the flagella of most motile bacteria in the intestine. Flagellin is recognized and bound by TLR5 on the cell membranes of immune cells, resulting in activation of these cells (218). Bacterial flagellin acts as a pathogen-associated molecular pattern (PAMP) with strong antigenicity, and plays a significant role under conditions of gut damage, including in inflammatory bowel disease (218–220), necrotizing enterocolitis (221), and diarrheal diseases (222, 223). Flagellin is considered to be the major immune antigen in Crohn’s disease, with bacterial flagellin antibody detected in approximately half of Crohn’s disease patients (224, 225). Anti-flagellin antibodies recognize the TLR5 and PATJ (PALS-1-associated tight junction protein) and induce monocyte activation and increased intestinal permeability in Crohn’s disease (226). Svärd et al., reported that circulating flagellin correlates with anti-flagellin levels, and is associated with activation of monocytes in chronic HIV-1-infected individuals (227).

Flagellin promotes virus entry into epithelial cells (228), and enhances HIV-1 induced mucosal immunity in the intestine (229). In vitro, the flagellin/TLR5 complex has been observed to directly trigger viral replication in HIV-infected cells (100). Thibault and colleagues have reported that flagellin, by itself, is able to activate latent HIV-1 provirus in T-lymphoid cells and provoke virus gene expression in central memory CD4⁺ T-cells (230). A study by Brichacek et al., showed that flagellin enhances HIV-1 replication, and activation of CD4⁺ T-cells in tonsillar tissue ex vivo (231). In PLWH, elevated levels of anti-flagellin immunoglobulin G (IgG) are present in ART-naïve HIV infected individuals, as compared to HIV-negative controls, and after ART treatment, the level of anti-flagellin IgG decreases (100, 232, 233). The outcomes of these studies imply that in PLWH, flagellin may accelerate the depletion of immune cells in the intestinal mucosa, which in turn worsens the integrity of the intestinal barrier and further encourages the entry of flagellin into the circulation.

D-lactate (D-LA) is a product released by bacteria residing in the human gut, and is not known to be produced in other tissues; thus, the D-LA present in plasma can be assumed to originate from the gastrointestinal tract (234–237). In other words, plasma D-LA levels may also be used as a surrogate for the degree of gut permeability and epithelial damage.

Increased plasma D-LA and LPS levels have been observed in some disease models that lead to intestinal mucosal damage. Increased D-LA levels are associated with diseases which induce severe intestinal injury (238–240). Cai et al., observed and reported on the correlation between D-LA and Crohn’s disease activity. Their study found that serum D-LA levels in patients with active Crohn’s disease and that of those in disease remission were 16.08 ± 4.8 mg/L and 11.16 ± 3.17 mg/L, respectively. Serum D-lactate levels were significantly higher in the active phase compared to the remission phase of Crohn’s disease (241). Teng et al., observed that serum D-LA is associated with acute gastrointestinal injury (AGI) and failure in critically ill patients. D-LA and LPS levels were observed to be higher in patients in the gastrointestinal dysfunction (GID) group or the gastrointestinal failure (GIF) group, than in the healthy control group (101).

Similarly, plasma D-LA can also be used as a marker to predict the degree of damage to the intestinal barrier in HIV patients, and D-LA is associated with recovery of CD4⁺ T-cell counts. HIV-positive individuals who are immunological non-responders despite ART are prone to malnutrition and compromised gut barriers, which further exacerbates chronic immune activation and inflammation (242). Geng et al., conducted enteral nutritional intervention in these populations and found that serum D-LA and LPS levels were significantly lower in PLWH, with good immune reconstitution after intervention, compared with levels of these markers pre-intervention. Also, D-LA levels negatively correlate with the recovery of CD4⁺ T-cells, and positively correlate with levels of the inflammatory factor, IL-1β (243).