The metabolic consequences of HIV/TB co-infection

People living with the human immunodeficiency virus/acquired immunodeficiency syndrome (PLWHA) have a 26–31 times greater risk of developing active tuberculosis (TB) compared to human immunodeficiency virus (HIV)-negative individuals [1] due to the immunosuppressive effects of HIV on the host. TB is the most common secondary disease among PLWHA, driving the global burden of HIV/TB co-infection, which has been exacerbated by diminished access to health care facilities during the coronavirus disease 2019 (COVID-19) pandemic. This resulted in poor diagnosis, reporting, and treatment initiation for HIV and TB cases, increasing TB-related deaths during this period [2‐4]. Approximately 12% of the global estimated 1.6 million TB-related deaths in 2021 were among PLWHA [5].

HIV (all references to “HIV” imply HIV-1) and Mycobacterium tuberculosis (Mtb) act synergistically during co-infection of a host, amplifying the disease burden. This synergy is centered on immunological deterioration [6, 7], which is the result of an increased level of immune activation [8] and oxidative stress (OS) [9]. The immune and metabolic systems are intricately connected, and changes in one are reflected in the other. While the host undergoes metabolic changes as part of immune processes, the pathogen modulates and/or reprograms host metabolism to suit its own needs, for example, reproduction and immune evasion or persistence [10]. This leads to metabolic dysfunction in the host, a condition that becomes particularly prominent if the immune response fails to eliminate the pathogen and the infection becomes chronic. This is the result of a continuously active immune response, forcing the host to adapt to long periods of higher energy and biosynthetic demand [11].

Metabolic alterations during co-infection are not well characterized. However, conventional techniques relying on low-throughput and laborious biochemical assays [12] have shown an impaired net protein balance [13] and hypoalbuminemia [14], along with similar body composition changes [15] in co-infected individuals compared to individuals with HIV infection or TB alone. Since these techniques do not provide a comprehensive picture of the metabolic state, metabolomics approaches would greatly contribute to the existing knowledge in the field [12]. Although metabolomics has been used to better understand specific aspects of host metabolism during co-infection, such as tryptophan metabolism [16‐18], an explorative approach to characterize the metabolic alterations that accompany HIV/TB co-infection has not been used. Untargeted metabolomics aims to characterize the metabolome holistically by identifying and quantifying as many metabolites as possible in a sample. It is also considered a hypothesis-generating research approach [19] which guides more focused or targeted research later. Therefore, untargeted metabolomics is ideal to provide a preliminary overview of the altered host metabolism during HIV/TB co-infection. From this, possible metabolic mechanisms associated with the HIV/TB synergy can be identified which will highlight areas of interest for future studies.

The altered gut microbiome caused by HIV infection [20] and TB [21] have been reported previously, although not well described or understood, and could play a role in host metabolism [22]. The metabolic effects of an altered gut microbiome linked to HIV/TB co-infection have not yet been investigated, however, this would be considered an important research interest considering the morphological changes previously reported in the intestines of co-infected patients [23]. Given the systemic nature of HIV infection and TB, as well as the potential involvement of microbial metabolites, it is preferable to be able to detect as many metabolites as possible. For this, two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) is ideal. The advantages of this technique include increased peak capacity, resolution, and sensitivity [24].

While antiretroviral therapy (ART) typically leads to decreased viral load, low-level inflammation persists, and metabolic complications are common in PLWHA [25]. The metabolic effects of ART in the context of HIV/TB co-infection has been investigated using conventional techniques (reviewed by Jain et al. [25]), and with a focus on the immune reconstitution inflammatory syndrome, using metabolomics [26]. Jain et al. [25] indicated that the metabolic complications experienced by patients vary even among those taking different antiretroviral drugs from the same class. The authors attributed this varied response to environmental and genetic factors, as well as duration of infection. However, to date, metabolomics has not been used to compare untreated and treated HIV/TB co-infection.

In this investigation, serum samples were collected from healthy controls, as well as patients with TB, and HIV/TB co-infection, with and without ART (all these individuals were untreated for TB). Since the sample groups were small, non-homogenous (in terms of the extent of disease and duration of ART) and lacking in associated clinical indicators (such as viral load in most cases), the statistical power would be too low to identify specific metabolites that could be investigated as accurate markers of, or therapeutic targets for, the HIV/TB co-infected state. Hence, metabolomics was used in a solely explorative manner as a first step for future studies. The aim was to identify areas of metabolism, or classes of metabolites, that could potentially be investigated in more detail in future studies, and to get an indication of how the metabolomics results would compare to what is already known. Given the inherent complexity of HIV infection and TB, multidisciplinary approaches such as metabolomics which incorporate several disciplines to address a research question, are crucial to understanding the mechanisms behind these infections. Including metabolomics techniques would therefore be highly informative, but requires some preliminary information, such as that provided here. As metabolic information could be useful to researchers investigating the co-infected state from perspectives other than metabolomics as well, these preliminary results and the ideas generated thereby, are explored here in the context of existing literature. In a previously published review, we generated hypotheses regarding the metabolic profile of HIV/TB co-infected patients, measurable by metabolomics [12]. Here, we expand on those hypotheses using data generated from the above-mentioned samples. These samples were analyzed using two-dimensional gas chromatography time-of-flight mass spectrometry to get as wide a view of the metabolism as possible.

A total of 529 compounds were detected in this study before data clean-up. Several statistically significant metabolites could not be correctly annotated, possibly due to low signal-to-noise ratios, unsuccessful deconvolution, or its absence from the libraries used [39]. Metabolites that could not be related to any biological function were labelled as “exogenous/unannotated metabolites” and are not discussed, thus their presence and function in human serum requires further investigation. Instances where metabolites were annotated with the same name but with different unique masses and where the mass spectra could not be visually matched, were listed separately, with the unique mass as identifier.

The metabolic profile associated with HIV/TB co-infection indicates significant overlap with TB alone, but also shows unique characteristics. These results highlight the significant amount of research that is still needed to fully characterize the human metabolome in general. The results were compared to existing literature to contextualize or explain the metabolic changes seen in this investigation.

Prior to addressing the aims of this study, a brief discussion will be provided on the metabolic changes associated with clinical and demographic parameters. In Fig. 3, metabolites related to lipid and protein metabolism (11,14-eicosadienoic acid, 2-(diethylamino)ethyl vaccenoate, and leucylleucine) were decreased, while mannitol was increased in the LCD group compared to the HCD group. This is expected, as individuals with a lower CD4 T-cell count would typically be at a more advanced stage of disease with higher viral loads. The greater severity of infection would place more strain on the host’s body as the virus uses more of the host resources for replication. Additionally, this might increase the success of Mtb in these individuals (see Kwan and Ernst [47] and Sharan et al. [7] and for reviews of the mechanisms by which this occurs), which would overwhelm the host’s metabolic system. Although highly plausible, this hypothesis cannot be confirmed in this study cohort since viral load and indicators of TB severity were unavailable. The decreased levels of 2-(diethylamino) ethyl vaccenoate in the LCD group implicates cholesterol metabolism as it is related to cholesterol ester cholesteryl 1-vaccenoate, which is a major constituent of lipoprotein particles. This is in accordance with previous results associating cholesterol and CD4 T-cell counts in untreated HIV infection [48] and the activation of lymphocytes in TB [49, 50]. Long-chain fatty acid metabolism is implicated by the decrease of 11,14-eicosadienoic acid in the LCD group, although not much is known about the role of this eicosadienoic acid derivative in human metabolism. It is interesting to note, the reduction of the dipeptide leucylleucine as one typically expects the opposite since protein breakdown products usually increase progressively during disease as the body starts using alternative fuel substrates to provide for the inflammation-driven elevated metabolic rate and energy demands. However, the reduction observed suggests that these individuals have already depleted their protein reservoirs, which supports the previously reported indications of an impaired net protein balance in co-infected individuals [13]. The increased levels of mannitol in the LCD group also supports this hypothesis. This increased mannitol may additionally indicate subclinical co-infections in the co-infected individuals and/or a more virulent microbiota composition [51, 52]. Gut inflammation has an impact on mannitol permeability in PLWHA [53], which could explain the increased mannitol observed in the LCD group, possibly indicating a more progressive phenotype. These metabolites, though detected in an extremely small cohort, give an indication of which metabolic analyses would be most pertinent to investigating the effect of CD4 T-cell count in individuals with HIV/TB co-infection. However, it is also clear from these and previous results that this will have to be done in combination with determining other biochemical parameters, body composition measurements, and importantly, viral load measurements, preferentially in a longitudinal study design.

Protein catabolism is a known metabolic consequence of HIV and TB infection [13, 14]. When exploring the data of the patient groups compared to the controls, the patient groups had comparatively elevated levels of 2-ketobutyric acid and L-proline and decreased levels of 4-hydroxyproline. This suggests the breakdown of proteins and amino acids for energy as these metabolites are intermediates of amino acid metabolism that feed into the tricarboxylic acid cycle. 3-Indolepropionic acid is another breakdown product of amino acids, specifically tryptophan, because of immune cell and microbial metabolism (this is explored in more detail below). Increased levels of glycerol (unique mass: 73 and 103) further indicate the use of alternative fuel substrates such as lipids, as a product of lipid hydrolysis. Furthermore, Mtb and other intracellular bacterial pathogens use glycerol-containing lipids as a fuel substrates [54]. For this, the bacteria synthesize their own phospholipases and rely on those of the host, such as phospholipase A2, which is released as part of the host’s inflammatory response [54, 55]. Arabinose was also significantly increased in the patient group and is an essential component of the Mtb cell wall [56] and has been associated with an altered gut microbiome as a result of COVID-19 [57] and other infections. Thus, the common metabolic profile of individuals with TB or HIV/TB co-infection suggests a state of low energy availability and an altered gut microbiome, possibly due to the chronic inflammatory nature of these diseases.

When investigating the data comparing the untreated HIV/TB co-infected group to that of untreated TB only (i.e., describing the effect of HIV infection on TB); phenylalanine was increased while tryptophan was reduced in the untreated co-infected group comparatively. Elevated phenylalanine has previously been reported in biofluids collected from untreated TB [36, 58‐61] and untreated PLWHA [43, 62, 63]. This can be ascribed to a decreased phenylalanine hydroxylase (PAH) activity [62, 63] due to a tetrahydrobiopterin (BH₄) deficiency [63] or compromised insulin secretion [36]. This likely occurs since the interferon-influenced BH₄ is sensitive to oxidation, aggravated during the high levels of OS experienced during infection [9, 64] in the co-infected group [65, 66]. Furthermore, macrophages and dendritic cells produce neopterin at the expense of BH₄, further depleting this, a mechanism confirmed by the fact that neopterin is known to be increased during both HIV infection [67] and TB [68], and is even more pronounced during HIV/TB co-infection [69]. Neopterin has also been shown to predict the development of active TB in PLWHA six months before a dramatic increase in viral load and reduction in CD4 T-cell count. Although neopterin was not detected as a metabolite marker in this study, the continued immune activation during HIV/TB co-infection [7] contributes to BH₄ depletion via neopterin overproduction, decreasing PAH activity. Thus, the increased phenylalanine levels may be ascribed to OS and immune activation by HIV infection.

Altered tryptophan metabolism via the kynurenine pathway as a result of increased indoleamine 2,3-dioxygenase 1 activity is a well-established consequence of various inflammatory conditions, including HIV [43‐45, 70‐72], TB [36, 58, 68, 73, 74] and HIV/TB co-infection [16, 17]. The indole products of tryptophan catabolism by the gut microbiota can be produced endogenously in an interleukin-4 inducible manner in T-cells and dendritic cells [75]. The importance of tryptophan catabolism by the gut microbiota during co-infection is reflected in the decreased levels of tryptophan and 3-indoleacetic acid (IAA) (as well as 3-indolepropionic acid [IPA] and 3-indolelactic acid [ILA] in the untreated HIV/TB co-infected group when compared to the healthy controls). Tryptophan was decreased in all patient groups relative to healthy controls, and this was most pronounced in the untreated co-infected group although not statistically significant (Table 2). Olsson et al. [18] recently showed tryptophan levels to be lower during HIV/TB co-infection when testing the suitability of the kynurenine/tryptophan ratio as a tool for the identification of TB cases in an untreated cohort of PLWHA. IPA, IAA, and ILA all have roles as antioxidants, ameliorate inflammation [76‐79], and regulate intestinal immunity by activating the aryl-hydrocarbon receptor transcription factor [75, 80]. Increased IPA has been associated with enhanced intestinal barrier integrity [81] and has mycobacterial-specific antibiotic activity [78], thus its decrease could relate to the loss of gut barrier integrity induced by HIV [82], intestinal damage known to occur in co-infected individuals [23], and possibly TB progression in PLWHA [78]. Typically, the catabolic products of tryptophan would increase as tryptophan decreases, however, the advanced disease state (as per CD4 T-cell counts in Table 1), high levels of OS [9, 64], and microbiome dysbiosis [20, 21] in the co-infected individuals may account for this observation. These data and previous reports confirm that HIV/TB co-infected individuals have a significantly increased tryptophan catabolism [16, 17] compared to individuals with either disease state alone, and suggests that the gut microbiota-derived tryptophan catabolites may be important indicators for the co-infected state. This could be used in conjunction with the kynurenine/tryptophan ratio, which has been suggested as a biomarker for co-infection [17, 18].

A general decrease in the lipid-related metabolites was observed in the untreated HIV/TB co-infected group relative to the untreated TB group, confirming previous reports of the effect of HIV on altering lipid metabolism [46]. Glycerol-3-phosphate is used for the rapid regeneration of reducing equivalents for oxidative phosphorylation [83] in the brain and skeletal muscle. The glycerol-3-phosphate shuttle also links fatty acid metabolism (which is typically dysregulated during HIV infection [84] and TB [85]) to glycolysis and oxidative phosphorylation [86], allowing fatty acids to be preferentially used for energy. Alpha-linolenic acid and arachidonic acid are essential polyunsaturated fatty acids (PUFAs) and are important structural components of cell membranes [87] and mediators of inflammation since they act as precursors to eicosanoids. Low levels of alpha-linolenic acid is associated with the failure to inactivate the virus during infection, resulting in virus proliferation and immunodeficiency [88]. PUFAs and glycerophospholipids are also important for distinguishing co-infected individuals who develop immune reconstitution inflammatory syndrome from those who do not [26]. Reduced levels of dodecanal (also called lauric aldehyde) were observed in the untreated co-infected group (Table S13), implicating decanoic acid metabolism [89] as an alternative energy substrate [31]. This fatty acid is typically produced as an intermediate of glycerophospholipid (specifically plasmalogens) metabolism and indicates changes in membrane turnover. It has previously been reported that plasmalogens are decreased during HIV infection [45, 90]. These lipids are essential to membrane structure and function, immunity, and serve as antioxidants to protect against lipid peroxidation [89]. Nonanamide and decamamide belong to the recently identified fatty amide sub-class of metabolites, which may have bioactive signaling functions [91]. Although not detected here, oleamide has been found to modulate the activity of 4-aminobutyric acid (GABA) receptors. Given the results discussed below, the effect of other fatty amides on signaling processes and metabolic enzymes requires further investigation, especially in HIV and its effect on TB in HIV/TB coinfected individuals.

The catabolism of GABA results in the production of 3,4-DHBA and 2,4-DHBA [92]. These hydroxy fatty acids were significantly increased in the untreated co-infected group relative to healthy controls — in fact, the untreated co-infected group had the highest levels compared to all other patient groups. 3,4-DHBA induces a feeling of satiety possibly due to its structural similarity to 3-hydroxybutyric acid [93], which is used as an alternative fuel substrate during fasting. These metabolites are also linked to poor overall health and nutritional status induced by an extremely low energy intake [94]. This suggests cachexia, which is in line with the overall indication of alternative energy substrate use and is characteristic of both HIV infection and TB [49, 95]. Elevated 3,4-DHBA has also been detected in the urine of unsuccessfully treated TB individuals [96], further confirming its association with a progressive phenotype. However, the exact mechanism of increased 3,4-DHBA and 2,4-DHBA concentrations have yet to be clarified. As such, 3,4-DHBA was subjected to correlation analysis with other significantly altered metabolites in this investigation to determine if this could yield new insights into the mechanisms of cachexia. 3,4-DHBA correlated positively with 2,4-DHBA, phenylalanine, 3-hydroxyisovaleric acid, and microbial metabolites (arabinose and rhamnose), while negatively correlating with tryptophan, IAA, threonine, 2-ketoisocaproic acid (another intermediate of leucine catabolism), and tetrahydrofuran. Therefore, given the mechanisms that result in the alteration of these correlating metabolites, 3,4-DHBA would increase with more pronounced inflammation and microbial translocation.

Microbiota metabolism is an essential component of human metabolism [22]. Phenylalanine and tryptophan metabolism by gut microbiota yields cresol and indole intermediates [97‐99]. While previously shown to be elevated during HIV infection [62, 100, 101], a p-cresol derivative, 2,4-di-tert-butyl-phenol (which cannot be produced by humans [102]), and benzoic acid [103] were reduced in the untreated HIV/TB co-infected group relative to those with TB alone. The impaired gut mucosal integrity, microbial translocation, and microbiome dysbiosis (which is also a feature of TB) induced by HIV infection [20, 21, 53] are generally reflected in the pathomorphological changes seen in the small intestine of HIV/TB co-infected individuals. Considering this, the decreased concentrations in the p-cresol derivative and benzoic acid in this study, induced by HIV infection in TB positive individuals, may result from an altered microbiota and increased OS [23] given the antioxidant properties of these metabolites. Furthermore, 2,4-di-tert-butyl-phenol has been detected in the fecal metabolome of COVID-19 patients and significantly correlated with specific populations of COVID-19-altered gut microbes [57]. Thus, its presence in serum may be due to translocation from the intestine, because of the HIV-associated ‘leaky gut’. Similarly, cadaverine, a microbially-produced antioxidant polyamine, was detected in reduced amounts in the untreated co-infected patients relative to the TB patients. This metabolite has been detected in HIV infection [62] and Mtb-related metabolomics studies [37, 104]. Since the gut microbiota produces cadaverine, the reduction observed here is most likely ascribed to the altered gut health and microbiota composition associated with HIV infection and TB [105]. Cadaverine forms part of the glutathione metabolic pathway, and as such, its reduction may be associated with OS [106]. Furthermore, reduced levels of glutathione with concomitantly increased indicators of lipid peroxidation has been reported in HIV/TB co-infected patients relative to patients with HIV infection only (with and without ART) [9], which supports exacerbated OS during HIV/TB co-infection.

Individuals on ART experience inflammation and immune activation, although to a lesser degree than before treatment [107]. Although ART did not generally alter the metabolic profile of the HIV/TB co-infected individuals to a statistically significant degree, the levels of alpha-linolenic acid, dodecanal and L-proline were significantly different and partially recovered (although not to the levels in healthy controls). This is in line with previous studies, which showed that ART does not restore the metabolic condition to that of healthy individuals, but rather induces further aberration [108] and still reflects continual inflammation despite viral suppression (reviewed by Hileman and Funderburg [107]). This is also reflected in the scores plot of the PLS-DA (Fig. 5B), which shows that the co-infected individuals on ART rather than the untreated co-infected individuals, lay furthest away from the healthy controls. 3-Hydroxyisovaleric acid was the only metabolite that was significantly different in co-infected individuals on ART that was not affected by HIV infection in addition to TB. Its increase may reflect the known effect of mitochondrial damage induced by ART (reviewed by Bañó et al. [109]) as it is a breakdown product of leucine. This may be a consequence of reduced methylcrotonyl-CoA carboxylase activity, as this starts a process that results in the release of the free 3-hydroxyisovaleric acid [110] but must be further investigated in this context. Figure 8 summarizes the potential effects of HIV and its treatment on HIV/TB co-infected individuals as discussed above.

HIV/TB co-infection results in a metabolic signature characteristic of what is typically seen in other infections i.e., elevated catabolism to meet the increased energy demand to fight infection. When the metabolic differences between comparative groups are analyzed more closely, the results indicate that HIV and its treatment augments TB synergistically, at least in part, through increased inflammation, OS, ART-induced mitochondrial damage, and its detrimental effects on gut health. These factors negatively impact energy availability, which weakens the host’s defenses and contributes to the increased frequency of poor outcomes for individuals with HIV/TB co-infection as opposed to those with TB alone. Although ART is typically associated with metabolic aberrations, these results indicate that it does reverse the trends caused by HIV infection for some metabolites. However, this reversal is not complete as it does not restore the metabolite levels of ART treated-PLWHA to that of healthy controls, and several other metabolites remained unchanged.

Considering the above, this investigation not only describes a metabolic signature of HIV/TB coinfection, but also gives clues to the disease mechanisms associated with HIV, TB, and the HIV/TB co-infected state. Furthermore, this study highlights those areas of metabolism most likely affected, for future investigations using more targeted metabolomics approaches in relevant biofluids. Besides the small sample size, another limitation of this study is the lack of samples from individuals receiving TB treatment with and without concomitant ART. Examples of possible approaches for future studies include an organic acid extraction and GC–MS, a semi-targeted lipid panel and LC–MS, and positive electrospray ionization mode and derivatization specific for the analyses of fatty acyl derivatives, a targeted acyl-carnitines study using LC–MS, as well as targeted nuclear magnetic resonance spectroscopy for sugars using biofluids from HIV/TB co-infected individuals — with and without treatment (anti-TB drugs and ART). Ideally, from the discussion it is evident that this should be combined with immunological and microbiota studies, as many of these metabolic changes are strongly associated with various immunological responses. Such studies would benefit from concomitant enzymatic assays to obtain more direct mechanistic information (not done here due to limited sample volumes). The host-microbe interactions, including that with communal microbes and the pathogens of interest (i.e., HIV and Mtb), at the metabolic level, especially in infectious diseases, is essential to fully understanding the immunometabolic aberrations experienced by the HIV- and Mtb-infected host. Such studies may also shed new light on the origins of and mechanisms that sustain the chronic immune activation and inflammation experienced during infection, even when virally suppressed though medication. These studies should ideally be done in a longitudinal study design, in cohorts that are well-characterized clinically and demographically. Furthermore, this study highlighted the need to better characterize the human metabolome, especially with reference to the identification of metabolites that occur in human biofluids but cannot be annotated, as well as those compounds that to date have no known biological function. The ability of metabolomics to distinguish individuals based on their health status was also shown in this investigation, with five supposedly healthy individuals clustering with the samples collected from those with the most advanced disease state. This occurrence would need to be elucidated further and would require metabolomics studies on a population scale to identify if metabolomics can be used to identify a subclinical diseased state that may be present in an individual not yet presenting with symptoms or active disease, for the purpose of early disease detection.