Factors regulating the differences in frequency of infiltration of Th17 and Treg of the blood–brain barrier

Controlling CD4+ immune cell infiltration of the brain is a leading aim in designing therapeutic strategies for a range of neuropathological disorders such as multiple sclerosis, Alzheimer’s disease, and depression. CD4+ T cells are a highly heterogeneous and reprogrammable family, which includes various distinctive cell types such as Th17, Th1, and Treg cells. Interestingly Th17 and Treg cells share a related transcriptomic profile, where the TGFβ–SMADS pathway plays a fundamental role in regulating the differentiation of both of these cell types. However, Th17 could be highly pathogenic and was shown to promote inflammation in various neuropathological disorders. Conversely, Treg is anti-inflammatory and is known to inhibit Th17. It could be noticed that Th17 frequencies of infiltration of the blood–brain barrier in various neurological disorders are significantly upregulated. However, Treg infiltration numbers are significantly low. The reasons behind these contradicting observations are still unknown. In this perspective, we propose that the difference in the T-cell receptor repertoire diversity, diapedesis pathways, chemokine expression, and mechanical properties of these two cell types could be contributing to answering this intriguing question.


Introduction
Understanding the mechanisms underlying CD4 + T-cell infiltration of the brain is crucial for developing effective treatments for neuroinflammatory disorders.Targeting specific subsets of CD4 + T cells may provide a more personalized treatment.Th17 cells, which constitute one of the main CD4 + T-cell subsets, are involved in neuroinflammation, while Treg cells play a protective role in suppressing excessive immune responses in the brain.Therefore, understanding the differences in their brain infiltration abilities could be a promising approach to developing effective therapies for inflammatory brain conditions.

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anti-inflammatory Treg-like cells (Tr1), which can lower central nervous system (CNS) inflammation (Fig. 1B) (Jankovic et al. 2010).Moreover, intermediate stable cell types such as RORγt + FoxP3 + IL17 − have also been identified (Kim et al. 2017).Taken together, current research indicates that Th17 and Treg cells are highly related, although they have different functions and different phenotypes.
The reasons behind the differences in the abilities of Th17 and Treg cells to infiltrate the blood-brain barrier (BBB) are not fully understood.Th17 cell infiltration of the brain in neurodegenerative diseases and psychological diseases is higher than that of Treg (Beurel and Lowell 2018;Dos et al. 2016) (Schneider-Hohendorf et al. 2010).Treg isolated from healthy individuals or homeostatic mouse models present higher migration abilities compared to Th17 cells.However, this transmigratory ability is diminished in neuropathological conditions such as multiple sclerosis (Schneider-Hohendorf et al. 2010).One of the main explanations put forward is that Th17 cell infiltration of the brain is due to the BBB damage incurred in the first stages of various neurological conditions such as multiple sclerosis (Balasa et al. 2021).However, this hypothesis does not explain why Treg cells cannot infiltrate the brain at a similar rate to that of Th17 cells.Our investigation showed a clear difference in frequency between Th17 and Treg cell migration from the periphery to the brain across the BBB in a model of depression caused by gut inflammation (Mickael et al. 2022).In particular, we found that naïve CD4 + T cells that have differentiated into pathogenic Th17 cells expressing IL17 and IFNγ have a unique ability to infiltrate the brain in comparison to Treg cells.Once inside the brain, we found that Th17 cells are associated with over-activation of astrocytes, demyelination, and the appearance of lesion-like structures.Our MRI studies of inflamed mice brains did not show significant BBB damage prior to CD4 + Th17 infiltration, thus supporting the hypothesis that Th17 possess specialized abilities that allow them to better infiltrate the brain compared to Tregs.However, the reasons behind these observations are not yet known.In the next section, we discuss four mechanisms that could be contributing to this difference.

Difference in the diapedesis route
During homeostasis, the brain is almost isolated, with a limited number of CD4 + T cells gaining access to it.During pathological conditions, CD4 + T cells infiltrate the brain through four stages (Kubick et al. 2020;Muller 2011).In the first stage, CD4 + T cells are captured from the bloodstream through the interaction between vascular cell adhesion molecule 1 (VCAM1) expressed on endothelial cells and α4β1 expressed on CD4 + T cells (Fig. 2A) (Muller 2011).In the second stage, the process of infiltration is activated through the interaction between C-C chemokine receptor type 7 (CCR7) expressed on T cells and chemokine (C-C motif) ligand 21 (CCL21) produced by the BBB endothelial cells (Kubick et al. 2020).In the third stage, the adhesion of CD4 + T cells to the BBB is initiated through the interaction between lymphocyte function-associated antigen 1 (LFA1) expressed on CD4 + T cells and ICAM1 (intercellular adhesion molecule 1).In the final stage, diapedesis starts with one of two pathways: (Basu et al. 2013) paracellular transmigration or (Beurel and Lowell 2018) transcellular transmigration (Fig. 2B).The clustering of ICAM1 and VCAM1 with their respective ligands could take the form of transmigratory cups.Transmigratory cups surround the lower area of the lymphocytes in both paracellular and transcellular migration (Muller 2011).However, their role is still controversial, and it is not known whether they affect the Th17/Treg axis.In addition, ICAM1/VCAM1 interaction with their respective ligands leads to a rise in calcium.Free calcium activates myosin light-chain kinase and initiates actin-myosin contraction (Winger et al. 2014).ICAM1 also activates the RhoA pathway, which activates myosin light chain kinase (MLCK) and hence actin contraction.In paracellular diapedesis, the adherens junctions are disassembled starting with the phosphorylation of VE-cadherin by protooncogene tyrosine-protein kinase (Src) and protein tyrosine kinase 2 beta (PyK2) through an ICAM1-mediated pathway.This process leads to the unbinding of β-catenin to VE-cadherin.In addition, RAC1 is activated through a VCAM1 pathway leading to VE-cadherin phosphorylation (Muller 2016).Several components known to play a role in increasing BBB adhesion (e.g., CD99 and JAM-A but not VE-cadherin) are transferred to a vesicle-like structure called the lateral border recycling compartment.Following that, PECAM1 facilitates the motion of CD4 + T cells (Wimmer et al. 2019).Conversely, in transcellular migration, CAV1 and CAV2 (i.e., caveolins) interact together to form an area that allows the CD4 + T cells to pass through the cell membrane (Mickael et al. 2021).Autoreactive Th17 cells produce IL17 and IL22, which disrupt tight junction proteins in the CNS endothelial cells in multiple sclerosis (Jadidi-Niaragh and Mirshafiey 2011).Interestingly, Th17 has been shown to infiltrate endothelial cells through a paracellular pathway (Fig. 2B) (Wimmer et al. 2019).Treg cells have been demonstrated to prefer the transcellular route in analogous systems, such as the human hepatic sinusoidal endothelium.Interestingly, CD4 + Th1 also prefers the transcellular route, mediated by the formation of caveolae (Fig. 2B) (Shetty et al. 2011).However, whether Treg cells follow a transcellular route to infiltrate the BBB is not yet known.Also, it is important to note that when platelet and endothelial cell adhesion molecule 1 (PECAM1) was knocked out, Th17 cells followed a transcellular pathway (Wimmer et al. 2019).Whether PECAM1 expression can influence Treg diapedesis is not yet known.Taken together, current reports suggest that there could be a difference in the favored migration route between Th17 and Treg cells.

Difference in TCR repertoire components
Methodically investigating the T-cell receptor (TCR) repertoires of Th17 and Treg cells could shed light on the factors leading to the difference in their infiltrative abilities.The TCR-CD3 unit is the main protein complex responsible for antigen recognition by CD4 + T cells through interacting with the MHCII protein-antigen complex expressed on the APCs (antigen-presenting cells) (Abbas et al. 2018).The TCR-CD3 complex is built of two variable chains, namely, α and β forming the TCR domain, connected with three CD3s (clusters of differentiation 3) transduction molecules (e.g., CD3δ/ε, CD3γ/ε, and CD3ζ/ζ) (Fig. 3A).The variable regions in the α and the β chains are controlled by the expression of the CDR3 sequence (Fig. 3B).In addition to other APCs, MHCII is expressed by the endothelial cells of the BBB (Lopes Pinheiro et al. 2016).In rheumatoid arthritis (RA), where an unbalance between Th17 and Treg has been shown to influence the disease outcome, it has been revealed that the diversity of Th17 was less in RA than in healthy controls (Jiang et al. 2019).The case was reversed among Treg cells' repertoire, indicating higher clonality among Th17 but not within Treg cells.The TCR repertoire has been recently investigated in the context of multiple sclerosis.A high correlation between TCR repertoire and viral antigen was reported (Amoriello et al. 2020).Thus, we hypothesize that the similarity between antigens recognized by Th17 cells' CDR3 sequences and peptides expressed on or presented by the endothelial cells of the BBB could be a leading factor in Th17 cell-specific recruitment to the brain.However, the difference between the Th17 and Treg repertoires has not yet been investigated.Innovative techniques such as single-cell RNA sequencing could shed more light on this intriguing question (Kubick et al. 2020).

Difference in chemokine receptor expression
Previous research has shown that CD4 + T cells migrating to the brain utilize C-C motif chemokine receptor 6 (CCR6) and C-C motif chemokine receptor 7 (CCR7) to seek their ligands produced by endothelial cells such as chemokine (C-C motif) ligand 21 (CCL21) and chemokine (C-C motif) ligand 20 (CCL20) (Kubick et al. 2020;Li et al. 2013).However, the differences in the chemokine receptor expression between Th7 and Treg cells are not yet clear.For example, CCR9 drives the differentiation and migration of Th17.Conversely, CCR9 inhibits Treg function (Wang et al. 2010;Evans-Marin et al. 2015).CCR9 was shown to be expressed in the hippocampus, particularly in neurons and astrocytes (Liu et al. 2007).However, endothelial cells' expression of CCR9 has not been evaluated.Furthermore, its role in regulating Th17 and Treg cell infiltration of the brain is yet to be found.

Difference in physical structure
The difference in the physical structure between Th17 and Treg cells could be contributing to the difference in their braininfiltration capacity.Cells investigate their microenvironment and direct their movement based on the lowest possible strain energy needed (Voloshin 2020) (Mickael and Edwar 2012).CD4 + T cells determine their lowest strain energy state using their filopodia and lamellipodia (Voloshin 2020) (Mickael and Edwar 2012).In an interesting study, the authors studied the motility of Th17 in multiple sclerosis using a two-photon laser microscope in the subarachnoid space of the spinal cords of mice treated with the myelin oligodendrocyte glycoprotein peptide (Dusi et al. 2019).Interestingly, Th17 presented limited motility (Mickael and Edwar 2012).Even more, although when the authors blocked LFA1 integrin, the dynamics of Th17 cells were affected; however, the cell deformability and biomechanics, manifested by alterations in the filopodia and lamellipodia, were minimal (Dusi et al. 2019).These results point toward the possibility that LFA1 might not be playing a role in Th17 cells' strain energy calculation, although this is not true for other CD4 + subsets such as Th1 (Dusi et al. 2019).These findings raise unanswered questions about the genes controlling Th17 motility in comparison to Treg cells and whether physical structure contributes to increasing the frequency of Th17 infiltration of the brain in neuro-inflammatory pathologies.

Conclusion
Th17 and Treg cell infiltration of the BBB constitutes an unsolved riddle.First, these two cell types share important developmental pathways.They are also reprogrammable into various intermediate stable cells along the Th17/Treg axis.However, several differences seem to control their ability to infiltrate the brain.These differences include, but are not limited to, their favored diapedesis mechanism, their expression of chemokine receptors, their TCR repertoire, and their biomechanical properties.Investigating these differences is critical in designing therapeutics that aim to inhibit Th17 proinflammatory effects while enhancing Treg anti-inflammatory function.
Fig. 3 The TCR repertoire constitutes another aspect of the difference between Th17 and Treg cells.A The TCR-CD3 complex is expressed by CD4 + T cells; its main function is to recognize the antigens presented by the MHCII expressed by APCs.The TCR consists of two chains, namely, α and β; each chain contains a constant part and a highly variable part that is antigen unique.The CD3 complex is composed of two ε chains, a single δ chain, a single γ chain, and two ζ chains.Its main function is the transduction of the activation signal.
The MHCII complex is formed of two α and β chains.B To be able to recognize a large number of antigens, the genes forming the variable part of the TCR complex pass through several stages of gene rearrangements in the thymus resulting in a unique sequence formed of (V), diversity (D), joining (J) gene segments, and constant (C) segments in the case of the β chain and VJC in the case of the α chain.Differences in CDR3α and CD3Rβ sequences between Th17 and Treg could be another reason behind the differences in their behavior

Fig. 1
Fig. 1 Th17 and Treg cells share similar transcriptomic machinery.A TGFβ-SMAD pathway activates STAT3 that in turn activates RORc (the master regulator of Th17).TGFβ also induces FoxP3 activation through a SMAD-mediated pathway.B Th17 and Treg are highly reprogrammable and can change their phenotypes based on their microenvironment cytokine concentrations

Fig. 2
Fig. 2 Diapedesis pathway for CD4 + T cells infiltrating the brain.A Various CD4 + T-cell populations infiltrating the BBB share through three common stages, namely, capture, activation, and adhesion.B However, CD4 + T-cell populations differ in their preference for the pathways of infiltrating the endothelial cells, where Th17 cells favor