Study Characteristics

The search strategy yielded a total of 1025 research studies, as indicated in Fig. 1. Duplicates (n = 104) were removed, resulting in 921 studies. Thereafter, abstracts and titles were screened and a total of 773 studies were excluded. Of the remaining 148 studies, full-text articles were assessed, and an additional 126 were excluded as described in Fig. 1. Using the specified selection criteria, 22 fundamental studies were eligible for inclusion.

Quality Assessment of the Included Studies

The kappa was 1.00 which indicates perfect agreement [46] between the two raters as all studies were considered high quality, with clear descriptions of cause and expected outcomes, a control group, the use of multiple investigations (e.g., inflammation and pathway activation), and appropriate measurements and techniques to answer the research question (Supplementary Table 1).

Study Design of Various Tat Experiments

In the selected studies (n = 22), a variety of sample types were analysed. These included primary microvascular endothelial cells (bMVEC), primary astrocytes, several astrocytic cell lines (CRT-MG, HEB, U373 MG, SVGA, U-87, and A172), primary microglia, and primary neurons. The majority of studies investigated astrocytic cell lines n = 11; [23, 36, 37, 39,40,41, 47,48,49,50,51], followed by primary astrocytes n = 9 [21, 29, 32, 48,49,50, 52,53,54], primary microglia n = 3; [25,26,27], primary bMVEC n = 3 [32, 54, 55], primary neurons n = 1 [53], and primary neuronal/astrocyte co-cultures n = 1 [56] (Table 1).

To quantify cytokine/inflammatory markers, both protein levels and mRNA transcripts were measured. Various methods were employed for this purpose: ELISA (n = 12), a combination of ELISA and PCR (n = 8), and a combination of multiplex assay and PCR (n = 2).

Many studies (73%, n = 16/22) utilized Tat treatment on CNS cells. The remaining studies either employed cells transfected with HIV-1 positive Tat constructs (18%, n = 4/22) or used a combination of both approaches (9%, n = 2/22). Amongst the studies employing Tat treatment on CNS cells, some conducted dose-response and/or kinetics-based experiments to identify the most appropriate Tat treatment conditions for optimal cytokine expression levels. From these studies, only seven studies measured dose responses [21, 32, 36, 37, 48] (Table 1). Different Tat concentrations were used depending on cell type (primary cells vs cell lines), and for measuring protein and transcripts, various assays were employed such as ELISA and PCR.

In both primary cells and cell lines, a concentration range of 100 to 1000 ng/ml or 50–100 nM consistently produced the strongest inflammatory responses at both transcript and protein levels [21, 26, 36, 37, 48, 49] (Table 1). In kinetic-based experiments, both primary cells and cell lines displayed mixed results when measuring transcripts and proteins. For protein detection, optimal incubation times ranged from 8 to 48 h [26, 36]. Meanwhile, for transcript detection, the times varied between 6 and 24 h [36, 48]. Interestingly, some studies reported that primary cells can yield optimal cytokine expression results in as short as 5 min to 1 h [37, 49]. However, the specific response might vary depending on the cytokine [26, 49]. Additionally, optimal inflammatory responses might be influenced significantly by incubation times. It is noteworthy that primary cells show heightened sensitivity compared to cell lines. As such, when using primary cells, lower concentrations and shorter incubation times with Tat might be sufficient to achieve an optimal inflammatory response.

We conducted an evaluation of the Tat length utilized in studies, as it is regarded as a significant factor in Tat’s functioning. Amongst all the studies reviewed, n = 6/22 (27%) did not specify the Tat length utilized. The predominant choice was Tat72, with n = 8/22 (36%) studies using it, followed by Tat86 utilized in n = 3/22 (14%) studies, and Tat101 utilized in n = 3/22 (14%) studies. Additionally, one study (n = 1/22, 5%) employed a combination of both Tat86 and Tat101 for transfection and treatment studies, whilst another utilized a Tat peptide 24–38 (n = 1/22, 5%) (Table 1).

Tat Induction and Immune Marker Levels

Studies investigated a broad spectrum of markers, including CCL2, CCL3, CCL4, CCL5, CXCL10, IL-1β, IL-6, IL-8, MMP-1, MMP-9, RANTES, TNF-α, and uPA. Amongst these, the most frequently examined markers were CCL2 (n = 10/22, 45%), TNF-α (n = 10/22, 45%), CCL5 (n = 7/22, 32%), IL-6 (n = 7/22, 32%), CXCL10 (n = 6/22, 27%), IL-8 (n = 6/22, 27%), and IL-1β (n = 6/22, 27%) (Table 1).

Whilst it is widely established that Tat can directly impact inflammation, there remains uncertainty regarding a consensus on the direction of its association with inflammation levels. Specifically, it is unclear whether Tat presence consistently leads to an increase in the levels of specific inflammatory markers compared to untreated controls across different experiments. To address this, we focused on the most frequently investigated inflammatory markers (chemokines, cytokines, and interleukins) in this field of research to determine if Tat consistently elevates the levels of these inflammatory markers.

Given that certain markers were studied more frequently, naturally accumulating more supporting evidence, we considered the frequency of investigation when interpreting the findings. In this review, we applied a criterion to identify markers as “noteworthy”, building upon previous approaches [57, 58]. A marker (whether a gene transcript or protein) was considered noteworthy if it met two criteria: (1) it was investigated in three or more independent studies (as shown by the red cut-offline in Fig. 2A) and (2) 75% or more of the studies that examined this marker reported consistent levels when treated or transfected with Tat. If both conditions were met, the marker was deemed a “noteworthy marker” of finding for subsequent research (Fig. 2A). The markers, CCL2, CCL3, CCL4, CCL5, CXCL10, IL-1β, IL-6, IL-8, and TNF-α were investigated by ≥ 3 independent studies, therefore meeting our first criteria (Fig. 2A). CCL2 (10/10, 100%), IL-6 (7/7, 100%), and IL-8 (7/8, 88%) levels in primary cells and/or cell lines were consistently higher in the presence of Tat thus satisfying criteria two. In contrast, whilst reported in ≥ 75% of studies, CCL4 (3/4, 75%) was not detected with Tat presence, thus also satisfying criteria 2 as a noteworthy finding. The levels of CCL3, CCL5, CXCL10, IL-1β, and TNF-α were inconsistent with Tat treatment, thus not meeting our criterion for noteworthy markers. These findings suggest that CCL2, IL-6, and IL-8 may primarily serve as targets of Tat-induced neuroinflammation. For these findings, we did not consider the potential influence of Tat length, as not all studies reported the specific Tat length utilized (n = 6, 28% not reported). Furthermore, when attempting to stratify groups according to Tat lengths, there were too few studies available for meaningful comparison.

Fig. 2

A The frequency of inflammatory markers investigated across studies investigating primary cells and cell lines. The inflammatory markers here are ordered as chemokines, interleukins, matrix metalloproteinase, cytokines, and urokinase. B The frequency of inflammatory markers investigated across studies investigating primary cells only. The inflammatory markers here are ordered as chemokines, interleukins, matrix metalloproteinase, cytokines, and urokinase

Considering that inflammatory responses may vary between primary cells and cell lines; we categorized the studies based on primary cells alone to assess whether these markers exhibited consistent levels in response to Tat treatment. Amongst the investigated markers, namely CCL2, CCL3 CCL5, IL-8, and TNF-α were examined by three or more independent studies, meeting our first criterion (Fig. 2B—red cut-offline).

In primary cells, CCL2 (6/6, 100%), IL-8 (3/4, 75%), and TNF-α (5/6, 83%) consistently displayed elevated levels in the presence of Tat, as reported in ≥ 75% of the investigations, meeting the second criteria for noteworthy markers induced by primary cells. Conversely, CCL3 (2/4, 50%) and CCL5 (2/4, 50%) were inconsistent in their detection with Tat presence in primary cells.

Thus, when considering both primary cells and cell lines together, Tat consistently induced higher levels of CCL2, IL-6, and IL-8, as measured by protein and transcript levels. However, when focusing exclusively on primary cells, a consistent elevation was observed for protein/transcripts for CCL2, IL-8, and TNF-α in response to Tat treatment. Across both cell types, higher levels of CCL2 and IL-8 were observed.

Pathways for Tat-Induced Neuroinflammation

Once HIV crosses the BBB, infected cells release the Tat protein (Fig. 3). The Tat protein can interact with cell various types to induce neuroinflammation (Fig. 3). From all the included studies, n = 13/22 studies also investigated the pathways involved in Tat-induced neuroinflammation. These included the pathways for the expression of CCL2 (5/12, 41%), CXCL10 (5/12, 41%), TNF-α (5/12, 41%), IL-6 (5/12, 41%), IL-8 (4/12, 33%), CCL5 (3/12, 25%), IL-1β (3/12, 25%), and at least one study (1/12, 8%) investigating CCL3, CCL4 and MMP9 (Table 2). We also aimed to determine if there was a consensus between studies for the Tat-induced increase of the commonly investigated inflammatory markers. However, fewer studies investigated pathways involved in Tat-induced neuroinflammation. Hence, we therefore looked at markers with findings from at least two independent studies. Collectively, studies reported that CXCL10 was mediated by the extracellular-signal regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and the p38 MAPK pathway [25, 48, 50, 51]. CCL2 was mediated by the activation of the ERK1/2 MAPK pathway, the PI3K pathway, and nuclear factor-kB (NF-kB) [25, 29, 48, 51, 52]. TNF expression was NF-kappa B-dependent [23, 27, 36, 37, 41]. The findings for CCL5 were mixed, with Nookala and colleagues suggesting that CCL5 was mediated by the JAK, PI3K/Akt, and p38 MAPK signalling pathways and NF-κB, AP-1, C/EBPα, and C/EBPγ transcription factors, whereas Aversa and colleagues suggested that the CCL5 was not mediated by the ERK1/2 MAPK pathway, the PI3K pathway, and nuclear factor-kB (NF-kB). Studies collectively showed the involvement of the p38 MAPK pathway for mediation of IL-8, and IL-6 [25, 27, 36, 37, 39, 48, 51]. For IL-6 and IL-8, expression was mediated by NF-kB and AP-1 transcription factors; however, AP-1 was differentially activated for either cytokine. In the case of IL-6, p38δ activated AP-1, however, JNK was involved in AP-1 activation for IL-8 production. On the other hand, both PI3K/Akt and p38 MAPK (β subunit) were found to be involved in the activation of NF-κB that led to IL-6 and IL-8 production [39] (Fig. 4).

Fig. 3

Mechanisms of Tat-induced neuroinflammation. (1) HIV-1 infected monocytes can traverse the blood–brain barrier (BBB). (2) After successfully passing through the BBB, these monocytes initiate the activation of macrophages, which subsequently release viral proteins like the Tat protein. (3) The Tat protein also damages the BBB which allows an increased transmigration of cells into the central nervous system (CNS). This Tat protein, in turn, triggers responses in CNS, including (4) endothelia, (5) astrocytes, and (6) microglia, leading to the induction of neuroinflammation. The activated astrocytes and microglia, through various signalling pathways, induce the production of inflammatory markers. Tat can directly cause damage to (7) neuronal cells

Fig. 4

Pathway for Tat induction of inflammatory markers, CCL2, CXCL10, IL-6, and IL-8. Tat induces an inflammatory response in both primary astrocytic cells and various astrocytic cell lines. Exposure to Tat instigates the activation of MAPK kinases (MKKs), which subsequently phosphorylate and activate MAPKs through diverse mechanisms. Amongst the activated MAPKs are extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and the p38 pathway. Once these MAPKs are activated, transcription factors within the cell nucleus are also triggered. These activated transcription factors, including AP-1 and the NF-κB pathway, bind to specific DNA sequences within the promoter region of astrocytic cells. This binding event leads to the transcription of chemokines and cytokines such as CCL2, CXCL10, IL-6, and IL-8 markers. The heightened transcription of these markers results in increased levels of cytokines and chemokines within the cell

Tat Sequence Variation, Length, and Neuroinflammation

Most studies have not explored the effect of Tat sequence variation/diversity on neuroinflammation levels. As a result, this review cannot provide commentary on this specific aspect. However, Boven and colleagues used isolates from the brain tissue of participants with HIV-associated dementia (HAD) and non-dementia controls. These clones displayed several sequence variations within the Tat protein. Yet, when comparing HAD with non-HAD, there were no significant differences in CCL2 and IL-1β levels amongst the various Tat clones. The isolates in this study highlighted several amino acid sequence variants. It remains unclear if single-point variations influenced these outcomes since this aspect was not the study’s focus [47].

The study by Mayne and colleagues compared Tat amino acid sequence variations from patients in the Kenyan (subtype A) and Baltimore (subtype B) cohorts. However, they did not study these variants’ effects on neuroinflammation in cell cultures [49]. Mishra and colleagues examined the difference between subtype B and C Tat in inducing inflammation. When astrocytes were exposed to Tat subtypes B and C, the former induced higher CCL2 levels (p < 0.003). Moreover, upon transfecting astrocytes with different Tat expression vectors—namely, Tat B (C31) and Tat C (C31S)—a significant induction difference in CCL2 was observed between control cells and Tat B treatment (p < 0.0001). In contrast, no such significant difference was observed between vector control cells and Tat C treatment. This suggests that Tat C might have diminished inflammatory effect. Notably, this indicates that the reduction in CCL2 levels with Tat C (CS) might depend on the CS motif’s presence at position 31 [34]. The recent study by Gao and colleagues demonstrated that HEB astroglial cells transfected with pCDNA3.1-flag-Tat-B exhibited significantly higher activity compared to those transfected with pCDNA3.1-flag-Tat-C. These findings suggest that subtype B Tat, rather than subtype C Tat, might interact with the extracellular domain of the NOTCH3 receptor. This interaction potentially triggers the NOTCH3 signalling pathway, which is associated with increased inflammation levels [23]. Collectively, these studies suggested that Tat sequence variation could play a role in influencing neuroinflammation in foundational studies [53]; however, this requires further investigation.

Furthermore, we aimed to evaluate whether the length of Tat utilized in the studies could impact neuroinflammation or the reporting of significant findings. We categorized studies according to Tat72, Tat86, and Tat101. A total of n = 8 studies utilized Tat72. From these studies, the findings were variable in that n = 5/8 (63%) displayed that at least one of the investigated markers within the respective studies was not detected after treatment/transfection. A total of n = 3 studies utilized Tat86. From these studies, the findings were variable in that n = 2/3 (66%) displayed that at least one of the investigated markers in the respective studies was not detected after treatment/transfection. This is in contrast to the n = 4 studies utilizing Tat101. All studies utilizing Tat 101 and investigated markers in these respective studies were detected after treatment/transfection (Table 1).

Direct and Indirect Tat-Induced Neuronal Apoptosis

Even though the primary focus of this review was not to determine Tat-induced neuronal apoptosis, we sought to determine if studies investigated the relationship between Tat-induced neuroinflammation and neuronal apoptosis. Only five studies measured direct and indirect cell viability due to Tat presence (Table 2). All studies reported that when Tat was applied to cells of the CNS, there was a significant increase in cell death within 24 h. In addition, using conditioned media exposed to Tat, neuronal apoptosis was also significantly increased, which suggests that Tat-induced neuroinflammation may be a contributor to the observed neuronal apoptosis (Table 2).