TLR2 Supports γδ T cell IL-17A Response to ocular surface commensals by Metabolic Reprogramming

The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival γδ T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival Vγ4 T cells through a combination of γδ TCR ligation and IL-1 signaling. Here, we identify Vγ6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that Vγ6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, IκBζ, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal γδ T cells.


INTRODUCTION
An immunological balance between microbes and mucosal barrier tissues is required to ensure tissue health and homeostasis.Specifically, immune cells must mount a response that limits the outgrowth of microbes from becoming pathobionts without causing immunopathology.
The cytokine interleukin (IL)-17 plays a critical role in this process.IL-17 is required at virtually every mucosal site in the body to limit outgrowth of bacteria and fungi in the steady-state (Li et al., 2019).Humans and mice deficient in production of IL-17A and F experience severe and lifethreatening infections (Acosta-Rodriguez et al., 2007).
Regulation of IL-17 production in CD4+ αβ T cells relies on a combination of T cell receptor stimulation and signals from proinflammatory cytokines like IL-23, IL-1β, and IL-6 (Gomez-Rodriguez et al., 2014;Zhou et al., 2007).Regulation of IL-17 production in unconventional lymphoid cells, such as invariant natural killer T cells (iNKT), innate lymphoid cells (ILC), and γδ T cells, which often have critical biological functions at mucosal barrier sites, is less well-understood.
Although IL-1β signaling is required by iNKT and γδ T cells for IL-17 production (Sutton et al., 2009), TCR ligation is essential for an optimal response (Duan et al., 2010).We as well as others have demonstrated the critical role of IL-17 produced by unconventional T cells in autoimmunity and mucosal host defense, highlighting the importance of mechanisms that govern the effector functions of these cells (Edwards et al., 2020;Sherlock et al., 2012;St Leger et al., 2018).
and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 1, 2024.; https://doi.org/10.1101/2024.04.01.587519The conjunctiva of the eye is a specialized mucosal tissue responsible for protecting the ocular surface against allergic and infectious insults (Knop and Knop, 2005).Mice deficient in IL-17A and IL-17F are highly susceptible to severe bacterial and fungal infections, including corneal Candida albicans infection and the spontaneous outgrowth of Staphylococcus spp.(Conti et al., 2014;St Leger et al., 2018).We previously demonstrated that conjunctival γδ T cells are key contributors to the protective IL-17 elicited at the ocular surface by the Grampositive commensal bacterium, Corynebacterium mastitidis (C.mast) (St Leger et al., 2017).In Vγ4 γδ T cells, IL-17A production relied on TCR ligation and IL-1β signaling.Notably, C. mast also stimulated IL-17A production in Vγ4-negative γδ T cells, but their identity and the signals they required to induce IL-17A were unclear.Tight control over the production of conjunctival IL-17A is required to both prevent the outgrowth of pathobionts, and to limit bystander damage to the ocular surface, such as occurs in dry eye and other ocular surface diseases (de Paiva et al., 2022;Gadjeva et al., 2010;Suryawanshi et al., 2011).Therefore, understanding how an eyecolonizing microbe stimulates IL-17 in the Vγ4-negative γδ T cell subset will be essential for deciphering how mucosal immune responses are maintained at the ocular surface.
IL-17 responses in γδ T cells can be stimulated indirectly by microbial components which are sensed by toll-like receptors (TLRs) on myeloid cells that produce IL-1β and IL-23, which in turn stimulate IL-17A-producing γδ Τ cells (γδΤ17) (Akira and Takeda, 2004).However, γδ Τ cells themselves express various TLRs, including TLR2, TLR4, TLR6 and TLR9, which can affect their effector functions.For example, direct TLR2 engagement in vivo by the synthetic LPS analog Pam3CSK4 enhances γδ T cell proliferation and IL-17 production (Marks et al., 2021;Martin et al., 2009;Reynolds et al., 2010).
In addition to immune activation pathways, the metabolic regulation of γδΤ17 cells has recently become an area of intense interest.The development and the pro-tumor function of γδΤ17 cells were shown to be under metabolic control (Cai et al., 2019;Lopes et al., 2021).
However, the molecular mechanisms and the role of TLR2 in metabolic programming of these cells are not known.Our study offers to bridge these gaps by elucidating how TLR2 signaling influences IL-17 production through metabolic reprogramming of C. mast-specific γδ T cells.
Utilizing TLR2-deficient (TLR2-/-) mice colonized with C. mast, we demonstrate that TLR2 expression is essential in both dendritic cells (DCs) and in γδ T cells themselves for optimal IL-17A production.Our findings reveal that intrinsic TLR2 signaling in γδ T cells is linked to activation of the transcription factor IκBζ and causes a metabolic shift to fatty acid oxidation to support IL-17 production.Our findings underscore the significance of TLR2 in the metabolic and immune regulatory pathways of mucosal γδ T cells and highlight its potential as a target for modulating immune responses at mucosal barriers.

TLR2 is required for IL-17A production in response to C. mast at the ocular surface
Ocular colonization with the microbe, C. mast, results in the expansion of γδΤ17 cells in the eye draining lymph nodes (DLNs) (St Leger et al., 2017).These cells accumulate in the conjunctiva where their functionality leads to a steady-state, non-pathogenic recruitment of neutrophils to the tissue.Even though TCR stimulation and IL-1β were shown to induce IL-17, the requirement of innate receptors in the production of IL-17 in these cells was still unresolved.
Therefore, we focused our attention on the requirement of TLR2 signaling in mucosal γδ T cell IL-17 responses.We ocularly colonized WT or TLR2 -/-mice with C. mast, as previously reported, and assessed the production of IL-17A in DLNs.We found the percentage of IL-17A positive cells and the mean fluorescence index (MFI) of IL-17A in DLNs were reduced in TLR2 -/-mice compared to WT mice (Fig. 1A -B).This was due, in part, to a reduction in proliferation as measured by the expression of Ki67 within DLN γδ T cells (Fig. 1C).There was a concurrent reduction of IL-17 in the γδ T cell compartment of the conjunctiva (Fig. 1D).The effect of the reduction in IL-17 was also observed in the recruitment of neutrophils to the conjunctiva (Fig. 1E).

TLR2 on γδ T cells is required for their IL-17A response to C. mast in vitro
TLR2 is expressed on γδ Τ cells and DCs at inflammatory sites (Mokuno et al., 2000;Oliveira-Nascimento et al., 2012) and is required for microbe-specific IL-17 production, we sought to identify the immune cells that express TLR2 in the context of ocular C. mast colonization.Within the conjunctiva, both DCs and γδ T cells express detectable levels of TLR2 after C. mast inoculation, suggesting a role for this receptor in C. mast-specific IL-17 response (Fig. 2A).In contrast, conjunctival αβ T cells did not express detectable levels of TLR2 regardless of the presence of C. mast.
These data prompted the question of whether TLR2 expression is necessary on DCs, γδ T cells, or both for C. mast-specific IL-17 response.To address this, we examined co-cultures of WT and TLR2-deficient DCs and gd T cells in all reciprocal combinations, in the presence or absence of heat-killed C. mast.Stimulation with C. mast expectedly enhanced IL-17A production in cultures containing WT DCs and WT γδ T cells.In contrast, TLR2 -/-DCs supported a significantly reduced gd IL-17 response: fewer IL-17A+ γδ T cells, lower MFI of IL-17-positive cells (Fig. S1A), and less secreted IL-17A as measured by ELISA (Fig. S1B).Because DCs activated by TLR2 signals secrete IL-1β and IL-23, needed for γδ T cells to express IL-17A, we measured the levels of these cytokines in the co-culture supernatants.TLR2 -/-DCs produced less IL-1β (but not IL-23) in response to C. mast stimulation (Fig. S1C-D), suggesting that diminished IL-1β production by TLR2 -/-DCs may have accounted for the reduced IL-17A production by γδ Τ cells.This was confirmed by the ability of exogenous IL-1β to restore IL-17A production to levels comparable to those observed with WT DCs (Fig. S1E).
Next, we examined the effect of TLR2 deficiency in γδ T cells in their response to C. mast.In the presence of WT DCs, TLR2 -/-γδ Τ cells still produced less IL-17A than WT, by flow cytometry and ELISA (Fig. 2B-C), despite undiminished IL-1β and IL-23 (Fig. 2D-E).This indicated that optimal IL-17A production in response to C. mast in these cultures required TLR2 expression on both DCs and γδ T cells.While the need for TLR2 on DCs can be explained by their production of IL-1β, endogenous TLR2 expression on γδ Τ cells is independently needed to support their IL-17 response.

The absence of TLR2 on γδ T cells decreases their IL-17A responses in vivo
To study the γδ cell-intrinsic role of TLR2 in vivo, we performed adoptive transfer of TLR2sufficient (WT) or deficient (KO) γδ T cells into TCRδ -/-mice, which lack γδ T cells entirely.Donor γδ Τ cells were purified by sorting from (C. mast-naïve) CD45.1 WT or CD45.2TLR2 -/- mice and were co-transferred at a 1:1 ratio to sublethally irradiated (450 R) TCRδ -/-recipient mice.One week after cell transfer, the recipients were ocularly associated with C. mast or PBS as a control, and after another week, we assessed their IL-17A responses (Fig. 3A).In the C. mast-unassociated group, both WT and TLR2 -/-γδ T cells populated the recipient mice with similar efficiency, indicating that TLR2 is not required for γδ Τ cell "fitness" over the observation time period (Fig. 3B).However, in mice associated with C. mast, TLR2 -/-γδ Τ cells displayed reduced responses compared to the co-transferred WT γδ T cells (Fig. 3B).We observed a lower percentage of IL-17A positive cells, diminished ex-vivo IL-17A production to PMA per cell (MFI), and a lower proportion of proliferating (Ki67 + ) cells (Fig. 3C-D).These results confirm the cell-intrinsic role of TLR2 on γδ T cells for activation and IL-17A production in response to C. mast in vivo.

TLR2 -/-Vγ6 cells exhibit a more profoundly impaired response to C. mast than Vγ4 cells
The function of γδ Τ cells in terms of cytokine production is closely linked to their usage of specific TCR repertoires.γδ T cell subsets expressing Vγ1, Vγ4, and Vγ6 (Heilig and Tonegawa's nomenclature) produce IL-17A (Heilig and Tonegawa, 1986;O'Brien and Born, 2020;Shibata et al., 2007).In our previous study, we identified IL-17A-producing Vγ4 cells at the ocular surface of C. mast-colonized mice (St Leger et al., 2017).We also observed a population of Vγ4-γδ T cells that produced IL-17 but did not characterize their TCR or their requirements for stimulation.We now noted that ocular C. mast association increased the number of Vγ1 -Vγ4 -Τ cells that produced IL-17A in the conjunctiva (Fig. 4A).Using a TCRspecific antibody (17D1), we identified them as expressing the Vγ6 TCR (Roark et al., 2004) (Fig. 4B).Analysis of eye DLNs revealed the presence of IL-17A + Vγ6 cells even in C. mastnaïve mice, but the number of these cells and their IL-17A-producing capacity were considerably increased after C. mast inoculation (Fig. 4C).Notably, expression of TLR2 became significantly elevated on Vγ6 γδ T cells after C. mast colonization compared to other populations of γδ T cells (Fig. 4D).
The elevated expression of TLR2 on Vγ6 γδ T cells raised the possibility that TLR2 may have divergent roles in Vγ4 and Vγ6 IL-17A-producing γδ T (γδ T17) cells.We therefore examined the kinetics of IL-17A production by Vγ4 and Vγ6 T cells in the DLNs in response to C. mast.
The response of Vγ6 cells occurred earlier and was stronger than that of Vγ4 cells, occurring by day 10 in WT mice (Fig. 4E-F) and TLR2 deficiency compromised IL-17A production and proliferation (Ki67) of Vγ6 T cells to a greater extent than that of Vγ4 T cells (Fig. 4G).Together, these data lead to the conclusion that Vγ6 T cells are more dependent on TLR2 signaling than are Vγ4 T cells for their responses to the eye-colonizing commensal.

TLR2 signals regulate the transcriptomic and epigenomic production of IL-17A in Vγ6 cells
To study the basis for the requirement of TLR2 signals in the Vγ6 T subset, we performed RNA-seq on Vγ6 cells sorted from mice prior to and after C. mast inoculation (Fig. S2A).The principal component analysis (PCA) plot of the transcriptome revealed distinct clusters formed by WT and TLR2 -/-Vγ6 γδ T cells (Fig. 5A).Consistent with observations presented in Figure 4, the Vγ6 Τ cells from TLR2 -/-mice displayed lower expression of Il17a -related genes, such as Il17a, Il17f, Il17ra, Rorc, and Ccr6 (Fig. 5B), confirming that the reduced production of IL-17A protein in TLR2 -/-Vγ6 γδ T cells is regulated at the transcriptional level.We also investigated the chromatin accessibility of Il17a loci in WT and TLR2 -/-Vγ6 cells by ATAC-seq.Two open chromatin regions (OCRs) were called in Il17a loci of Vγ6 cells (Fig. 5C).Based on their genomic location and literature, we parsed one OCR in the promoter region of Il17a gene and the other OCR located at the conserved cis-regulatory element 2 (CNS2) region approximately 5500bp upstream of Il17a transcriptional start site (Akimzhanov et al., 2007;Wang et al., 2012).
Comparing chromatin accessibility in Il17a loci of other IL-17A-producing cells, such as Vγ4 and ΙLC3 cells, as reported by ImmGen Consortium, we observed a prominent peak at the CNS2 region of their Il17a loci, confirming that this region serves as a conserved cis-regulatory element of Il17a loci in these IL-17A producing immune cells (Fig. S2B).Vγ6 cells from C. mast-associated WT mice displayed significantly higher accessibility in the Il17a CNS2 region than those from C. mast-associated TLR2 -/-mice (Fig. 5C).This finding corroborates that TLR2 signals regulate IL-17A production in Vγ6 cells at the transcriptomic and epigenomic levels.
To identify TLR2-mediated transcription factors (TFs) that influence accessibility of the Il17a CNS2 region, we screened TFs known to bind to the Il17a CNS2 region and compared their expression between WT and TLR2 deficient Vγ6 cells detected by RNAseq.Of particular interest to us was IκBζ (encoded by gene nfkbiz) because of its capacity to bind the il17a CNS2 locus in Th17 cells (Okamoto et al., 2010).Furthermore, its transcript levels correlated positively with TLR2 transcript expression quantified by RNAseq and real-time PCR (Fig. 5D).
To examine whether TLR2 activation increases the expression of IκBζ, which in turn promotes transcription of Il17a, we sorted Vγ6 cells from Vγ6 TCR-transgenic mice (Hayes et al., 2005), treated them with the TLR2 ligand Pam3CSK4 in vitro, and measured the gene expression of IκBζ and IL-17A.As expected, Vγ6 cells after Pam3CSK4 stimulation expressed more transcripts of both nfkbiz and Il17a compared to stimulation with IL-7 (Fig. 5E).Next, we generated IκBζdeficient Vγ6 cells using a Cas9/gRNA ribonucleoprotein (RNP) transfection that targeted nfkbiz (Fig. S2C).Compared to Vγ6 cells transfected with a non-targeting control, IκBζ-deficient Vγ6 cells had diminished Il17a transcription (Fig. 5F).These findings indicate that TLR2 in Vγ6 cells promotes IL-17A production at the transcriptomic and epigenomic levels, at least in part through the involvement of IκBζ.

TLR2-deficient Vγ6 T cells show impaired fatty acid oxidation
Ingenuity Pathway Analysis (IPA) of differentially expressed genes in C. mast-associated mice revealed that WT Vγ6 T cells were enriched in the Oxidative Phosphorylation (OXPHOS) pathway (Fig. 6A).OXPHOS takes place in mitochondria and generates ATP through oxidation of nutrients to meet cellular energy demands.We assessed the mitochondrial mass in Vγ6 T cells using MitoTracker Green staining.Both WT and TLR2 -/-Vγ6 cells displayed a similar increase in mitochondrial mass upon C. mast association, indicating TLR2 deficiency did not affect mitochondrial mass (Fig. S3A).The percentage of MitoSOX (mitochondria superoxide indicator)-positive cells was also comparable between TLR2 -/-Vγ6 cells and WT Vγ6 cells (Fig. S3B).In contrast, MitoTracker™ Red CMXros staining, which measures mitochondrial membrane potential (ΔΨm), was reduced in C. mast-associated TLR2 -/-Vγ6 cells (Fig. 6B), implying impaired mitochondrial activity.This was supported by a reduced ATP production in TLR2 -/-Vγ6 cells compared to WT Vγ6 cells (Fig. 6C).
T Cells can generate ATP through the OXPHOS and/or glycolysis pathways.To determine which ATP production pathway is affected in TLR2 -/-Vγ6 cells, we used metabolic flux analysis.
To obtain sufficient TLR2 -/-Vγ6 cells, we crossed Vγ6 TCR-transgenic mice (Hayes et al., 2005) onto a TLR2 -/-background (denoted as WT Vγ6 Tg or TLR2 -/-Vγ6 Tg, respectively).First, we wanted to verify that TLR2 -/-Vγ6 Tg (double mutant) cells recapitulated the phenotype of Vγ6 cells from TLR2 -/-mice with a conventional TCR repertoire.Stimulation with C. mast in the presence of WT DC confirmed that TLR2 -/-Vγ6 Tg cells had similarly deficient IL-17A and ATP production to TLR2 -/-Vγ6 cells from mice with a WT TCR repertoire (Fig. S3C).Next, we performed metabolic flux analysis comparing WT Vγ6 and TLR2 -/-Vγ6 Tg cells using a Seahorse assay.The mitochondrial stress test revealed a significant decrease in basic and maximal oxygen consumption rate (OCR) in the TLR2 -/-Vγ6 Tg cells compared to WT Vγ6 Tg cells (Fig. 6D-E).The cell-intrinsic role of TLR2 on OXPHOS was confirmed by direct Pam3CSK4 stimulation of Vγ6 cells in vitro (Fig. S3D).In contrast, the glycolytic proton efflux rate (glycoPER), which measures glycolysis, showed no differences between the two groups (Fig. S3E).Taken together, these findings indicated that, glycolysis was unaffected, but TLR2 -/-Vγ6 cells exhibited impaired OXPHOS, leading to insufficient ATP production in these cells.
ATP production in mitochondria mainly comes from three substrates: glucose, long-chain fatty acid (LCFA), and glutamine.To determine the dominant substrate(s) in the energy metabolism of Vγ6 cells, we performed the Substrate Oxidation Stress Test on Vγ6 Tg cells from TLR2sufficient Vγ6 Tg mice.Only etomoxir, an inhibitor of the LCFA oxidation pathway, significantly decreased both basic and maximal OCR in Vγ6 Tg cells (Fig. S3F), demonstrating that γδΤ17 cells utilize primarily fatty acid oxidation for energy production.To test whether TLR2 deficiency impairs the LCFA pathway in Vγ6 cells, we performed a Substrate Oxidation Stress Test on Vγ6 cells from WT Vγ6 Τg and TLR2 -/-Vγ6 Τg mice inoculated with C. mast.Vγ6 cells from TLR2 -/- Vγ6 Τg mice displayed a reduced basic and maximal OCR compared to WT Vγ6 Τg mice after etomoxir treatment, consistent with the reduced ATP content in TLR2 -/-Vγ6 cells (Fig. 6 F-G).
These data confirm that TLR2 signaling supports fatty acid oxidation (FAO) in Vγ6 cells.

Impaired Cpt1 function underlies the defective IL-17A response of TLR2-deficient Vγ6 cells
To further explore the underlying mechanisms, we studied the impact of TLR2 signaling on fatty acid oxidation (FAO) in Vγ6 γδ Τ cells.ATAC-seq data revealed that C. mast association activated an OCR within 2kb upstream of the Cpt1a gene, which encodes carnitine palmitoyl transferase 1a, a key enzyme involved in FAO (Fig. 7A).In addition, analysis of ImmGen Consortium data (Yoshida et al., 2019) identified the same OCR in the Cpt1a locus in IL-17Aproducing ILC3s from small intestine, which harbors commensal microbiota (Fig. S4).
and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 1, 2024.; https://doi.org/10.1101/2024.04.01.587519 doi: bioRxiv preprint Notably, TLR2 deficiency decreased chromatin accessibility at Cpt1a (Fig. 7A).These data suggest that TLR2 signaling can enhance FAO by promoting the transcription of Cpt1a, which is also supported by our in vitro data showing that Pam3CSK4 stimulation upregulated Cpt1a in Vγ6 cells (Fig. 7B).
We next examined directly whether FAO is involved in TLR2-mediated IL-17 response in Vγ6 cells.Treatment with etomoxir, an irreversible inhibitor of Cpt1a, blocked the upregulation of Il17a induced by TLR2 activation (Fig. 7C).Notably, IκBζ, which in our hands upregulates Il17a in Vγ6 cells in response to TLR2 signaling, had also been reported to upregulate Cpt1a in fibroblastic reticular cells (Majumder et al., 2019).In line with this, IκBζ-deficient Vγ6 cells generated by Cas9/Crispr displayed reduced Cpt1a transcription (Fig. 7D).In the aggregate, our data support a sequence of events where, in response to endogenous TLR2 signaling in Vγ6 cells, increased expression of IκBζ enhances transcription of Cpt1a, thus favoring fatty acid oxidation and providing metabolic support for IL-17A production.

DISCUSSION
Our earlier studies demonstrated that the ocular commensal C. mast elicits expansion and IL-17A production by γδ Τ cells that provide local defense from pathogens at the ocular surface mucosal barrier.The current study explores mechanistically how these γδ T cells sense and respond to C. mast.We demonstrate that C. mast association activates TLR2 signals on both DCs and γδ Τ cells.While engagement of TLR2 on DC triggered the IL-1β essential to elicit γδ T cell IL-17 production, intrinsic TLR2 signaling in γδ Τ cells was also required.Our data reveal that intrinsic TLR2 signaling in γδ17 T cells acts in a subset-specific manner to enhance transcription of genes related to IL-17A production and activates the metabolic reprogramming to fatty acid oxidation that is needed to support IL-17A production.
Microorganisms activate TLR2 signaling in antigen-presenting cells and Τ cells (both αβ and γδ Τ cells) for IL-17A responses (Reynolds et al., 2010).In our investigation of C. mast, we were able to dissect the contribution of TLR2 expression in DCs from that of γδ Τ cells.Specifically, where the presence of Vγ6, but not Vγ4, cells is dependent on the presence of commensals (O'Brien and Born, 2020;Wilharm et al., 2019).Based on our findings, we speculate that activation of innate receptors such as TLR2, by these commensals, may explain persistence in the gingival Vγ6, cells but not for Vγ4 cells.These phenotypes may perhaps be connected to the inherited innate-like behavior of Vγ6 cells towards C. mast and apparently also other commensals (Lu et al., 2015;Marchitto et al., 2019).Vγ4 cells, on the other hand, are laterstage responders, which may be explained by their need to receive TCR stimulation in addition to signals provided by DCs (St Leger et al., 2017).
TLR2 signals on Vγ6 cells target transcription factors in IL-17A regulation.We observed that TLR2 deficient Vγ6 cells displayed a decrease in the transcriptional expression of IκBζ.This transcription factor was found to upregulate Il17a in αβ Th17 cells (Okamoto et al., 2010).
However, the need for IκBζ in IL-17A production by γδT17 cells has not been reported, and in view of the very different developmental and functional niches of these lineages, was not a given.In our hands, an in vitro knockout of IκBζ reduced Il17a transcript levels in Vγ6 cells, suggesting a supportive role of IκBζ in Vγ6 cells' IL-17A pathways.In addition to transcriptional regulation, we observed the epigenomic regulation mediated by TLR2 signaling at Il17a locus in Vγ6 cells, especially at the promoter and CNS2 region.The transcription factor IκBζ binds to the Il17a CNS2 region in αβ Th17 cells, facilitating IL-17 transcription (Okamoto et al., 2010).
Although our study did not directly examine the binding of IκBζ to the Il17a regulatory region in Vγ6 cells, it stands to reason that lower binding of IκBζ to Il17a CNS2 region would contribute to the decreased Il17a transcripts in TLR2-deficienct Vγ6 cells.Notably, IκBζ does not act alone; it cooperates with Rorc to stimulate Il17a transcription in Th17 cells (Ciofani et al., 2012;Okamoto et al., 2010).Our RNA-sequencing data revealed a positive association between Rorc and TLR2 sufficiency, which supports the notion that Rorc upregulates Il17a through TLR2 signaling in Vγ6 cells.These findings suggest that TLR2 signaling utilizes multiple transcription factors that work together to regulate the transcriptional and epigenomic IL-17A program in Vγ6 cells.
Recent data made it clear that cell metabolism critically influences the functionality of immune cells.Cell metabolism is largely dictated by energy availability in the form of glucose, fatty acids, or other sources.However, other factors can impact cellular energy profiles.Here, we show that C. mast, through TLR2, can activate the OXPHOS pathway in γδ T cells, which then supports the production of IL-17A at the ocular surface.We posit that this finding parallels the situation found in the intestine, where colonizing segmented filamentous bacteria (SFB) can stimulate lipid metabolism in CD4 + Th17 cells (Omenetti et al., 2019).Similarly, type 3 innate lymphoid cells (ILC3s) produce IL-17A in a manner that correlates with increased lipid metabolism (Di Luccia et al., 2019).These results, together with observations that germ-free mice display impaired host metabolism and IL-17 responses at intestinal mucosal sites (Honda and Littman, 2016;Ivanov et al., 2009;Martin et al., 2009), suggest a shared metabolic regulation network across various mucosal surfaces that depends on the colonization of microbes at specific mucosal sites.

CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rachel Caspi (caspir@mail.nih.gov) and Xiaoyan Xu (xiaoyan.xu2@nih.gov

Data and Code availability
The RNA-seq datasets and the ATAC-seq datasets generated during this study are available at NCBI GEO (GSE236387).The code used for data analysis is shared at https://github.com/NIH-NEI/tlr2-ATAC-seq/

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice WT C57BL/6J mice were purchased from Jackson Laboratory.TLR2 -/-, TCRβ -/-, and CD45.1 C57BL/6J mice originally from Jackson were bred and maintained in our animal facility.Vγ6 transgenic mice were generously provided by Paul E. Love; TLR2 -/-Vγ6 transgenic mice, and TLR2 -/- TCRβ -/-mice were crossed and bred in house.For most in vivo and in vitro animal studies, male and female mice aged 6-10 weeks were used.All mice were housed under NIH SPF conditions.All mice received tear washes and were confirmed to lack ocular surface C. mast colonization before the experiment.All mouse studies were performed in full compliance with IACUC-approved protocols (NEI-690) and institutional guidelines.

Bacteria
C. mast was initially isolated from conjunctival homogenates that were plated on TSA + 5% blood agar plates kept at 37℃ in anaerobic conditions for 7 days.After original isolation, C. mast was propagated using TSA with 5% blood agar plates at 37℃ in an aerobic chamber (St Leger et al., 2017).Further, C. mast can be grown in blood heart infusion broth with 1% Tween 80 aerobically with shaking at 37℃.

METHOD DETAILS
C. mast Inoculation C. mast was applied (10 8 CFU) to the ocular surface every three days for a total of three inoculations.Three weeks after the final inoculation, 10 μl PBS per eye was used to wash the conjunctiva to confirm colonization.

Tissue harvest and processing
Mice were euthanized by extensive cardiac perfusion.Two conjunctivae of one mouse were harvested by excising the eyelid and bulbar conjunctiva, combined, minced, and exposed to 0.9 ml of 1 mM CaCl2, 2 mg/ml collagenase D, 0.16 mg/ml DNaseI and 0.05 mg/ml Dispase II in HBSS for 45 min at 37℃ with shaking.Cervical and submandibular lymph nodes were isolated, minced and exposed to 1mg/ml collagenase D for 30 min at 37℃ with shaking.After collagenase treatment, conjunctiva and draining lymph node cells were separately filtered through a 40 μm filter using a 1 cc syringe plunger.Then conjunctival cells were filtered a final time using a Corning Falcon Test Tube with Cell Strainer Snap Cap (Corning 352235).

CD11c Isolation
Mouse spleen tissue was cut into small pieces in RPMI medium containing 1mg/ml collagenase D. Tissue was incubated at 37°C and placed on a shaker for 1 hour.The single-cell suspension was prepared with a 70um filter and 1cc syringe plunger and was incubated with Miltenyi CD11c Isolation Beads and Fc Blocker for 10 mins on ice.CD11c positive selection was then performed following the manufacturer's protocol.

Flow Cytometry
In vitro assay: For intracellular staining, Brefeldin A (Golgi Plug) was added for the last 6 hours of 72-hour co-culture.After centrifugation, the supernatant was collected for ELISA assays.Cells were collected for flow cytometry.In vivo assay: tissue was homogenized in 2% FBS RPMI and filtered by a 40 μm strainer.The cells were stained following the manufacturer's protocol.For phospho-signal flow cytometry, we use BD Phosflow Fix Buffer and BD Phosflow Perm/Wash Buffer III.All the procedures followed by BD™ Phosflow Protocols for Mouse Splenocytes or Thymocytes.All samples were acquired with Beckman CytoFLEX LX Flow Cytometer and analyzed with FlowJo software.

RNA-seq
-cells were sorted from naïve WT, naïve TLR2 -/-, C. mast inoculated WT and C. mast inoculated TLR2 -/-mice.RNA was isolated using Qiagen miRNeasy Micro Kit.RNA was run on Agilent 2100 Bioanalyzer and RIN >8.0 was aimed to pass QC.RNA-seq was performed on Illumina Hiseq4000 (150pb, paired) using Clontech RNA Ultra Low Input Library Prep and pairedend sequencing in the NCI sequencing facility.Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt before alignment with the reference genome (Mouse -mm10) and the annotated transcripts using STAR.The mapping statistics were calculated using Picard software.
Library complexity is measured in terms of unique fragments in the mapped reads using Picard's MarkDuplicate utility.In addition, the gene expression quantification analysis was performed for all samples using STAR/RSEM tools.
ATAC-seq DAPI -CD90.2+ TCRβ -TCRγδ + Vγ1 -Vγ4 -CD27 -CD44 hi cells were sorted from naïve WT, naïve TLR2 -/-, C. mast inoculated WT, and C. mast inoculated TLR2 -/-mice.Library preparation and sequencing were done by Novogene based on a previously reported method (Corces et al., 2017).Briefly, the nuclei extracted from the cells were quality controlled and suspended in the TruePrep Tagment Enzyme mix and incubated at 37°C for 30 min, followed by PCR amplification and purification with the AMPure beads.The quality-controlled library was quantified with Qubit 2.0, clustered using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) and sequenced on an Illumina Hiseq platform to generate 150 bp pairedend reads.Raw data underwent rigorous quality control through FastqQC and FastP (Chen et al., 2018).Alignment was executed with Bowtie 2 (Langmead and Salzberg, 2012) and peaks were annotated using Genrich (Gaspar, 2021).ChiPseeker (Yu et al., 2015), and differential expression analysis relied on DESeq2 (Love et al., 2014).Homer2 was employed for motif analysis, and the UCSC genome browser (Karolchik et al., 2003) was the platform for peak visualization.

Mitochondrial ROS measurement
Cells were collected from PBS-treated and C. mast-treated WT and TLR2 -/-mice.Cells were incubated with a MitoSOX probe (Invitrogen, Cat.# M36008) for 10 min at 37℃.After incubation, cells were washed and stained for other surface markers.Then cells were subjected to flow cytometry analysis.
ATP level determination γδ T cells were sorted from C. mast inoculated WT and TLR2 -/-Vg6 transgenic mice and placed in 96 well, pre-coated with anti-TCRγδ (2μg/ml) the previous night.Cells were harvested 3 days post in vitro activation, washed with RPMI-1640 medium three times, and counted.Cells were rested in 96well for 3 hours at 37℃.Cells were quickly spun down and lysed in a native lysis buffer (Cell Signaling Technology) for 10 min on ice.Cells were then centrifuged at 2000 g for 10 min and the supernatants were collected.ATP levels were measured according to the manufacturer's instructions (Invitrogen, #A22066).

QUANTITATION AND STATISTICAL ANALYSIS
TLR2 activation in DCs by C. mast induced IL-1β, facilitating the proliferation and IL-17A production in γδ T cells.While both Vγ4 and Vγ6 cells exhibited IL-17A responses to C. mast, intrinsic TLR2 signals play a dominant role in Vγ6, but not Vγ4, cells for their proliferation and effector functions.The greater reliance on TLR2 signals for IL-17A responses in Vγ6 cells stimulated by C. mast are reminiscent of other reported phenotypes, e.g., in mouse gingiva, TLR2 signaling, functioning as a sensor of commensal bacteria, is involved in commensalmediated metabolic adaptation in γδ Τ17 cells.While previous studies have reported the impact of TLR2 signals on proliferation, survival, and cytokine production in various T cell populations, our findings expand the understanding of TLR2 signaling in the metabolic regulation of γδΤ17 cells.TLR2-activated FAO can increase expression of the Il17a gene.Mechanistically, TLR2 signaling can upregulate the expression of Cpt1a in Vγ6 cells, which correlates with enhanced FAO capacity.Notably, IκBζ, which increases Il17a transcription, also enhances the expression of Cpt1a in Vγ6 cells.These data suggest a possible feed-forward mechanism where factors downstream of TLR2 signaling can epigenetically influence the future identity and metabolic profile of the cell.The mechanism(s) through which IkBζ promotes Cpt1a expression require(s) further investigation.In summary, our results highlight the importance of intrinsic TLR2 signaling in γδ Τ cells to produce IL-17A in response to bacterial colonization.TLR2 signals enhance the expression of genes responsible for the IL-17A pathway and fatty acid metabolism in IL-17A-producing γδ Τ cells.Consequently, this study further links IL-17A production in γδ Τ cells with cell metabolism, implicating metabolic intervention as a potential strategy to modify γδ T cell functionality at mucosal and/or other barrier surfaces.

Figure 4 .
Figure 4. TLR2 -/-Vγ6 cells exhibit a more profoundly impaired response to C. mast than Vγ4 cells C. mast ( CFU =10 8 ) or PBS was instilled into each eye of C57BL/6J wild-type mice every 3 days for a total of 3 times.(A-D).(A)Representative FACS plots and bar graphs representing the number of IL-17A + Vγ1 -Vγ4 -γδ T in the conjunctiva of wild-type mice with/without C. mast inoculation.N= 6.Data were combined from 2 experiments.(B) Representative FACS plots showing the percentage of Vγ1 + , Vγ4 + , and Vγ6 + cells among IL-17A producing γδ T cells of eye-draining lymph nodes with or without C. mast inoculation.N= 3. (C) The percentage and the number of IL-17 + Vγ6 cells in eye-draining lymph nodes with or without C. mast inoculation.N= 4. (D) Representative FACS plots representing TLR2 expression in the indicated γδ Τ cell subset and bar graphs depicting normalized TLR2 mean fluorescein intensity (denoted as Δ MFI) of the indicated γδ subsets in eye draining lymph nodes.N=4.(Δ MFI = averaged MFI of TLR2 in C. mast + group -Averaged MFI of TLR2 in C. mast -group) (E-F) Kinetic experiment showing cell numbers of IL17A + Vγ4 + (E) and IL17A + Vγ6 + (F) from WT and TLR2 -/- mice with or without C. mast inoculation on the indicated days.N = 4. (G) Bar plot showing Ki-67 expression of γδ T cells in Vγ4 and Vγ6 subsets in WT and TLR2 -/-mice with/without C. mast inoculation.N=17.Combined data from 3 experiments.Bars represent mean ± SEM with *P<0.05,**P<0.01,***P <0.001.Statistical significance was determined by Welch's t-test (D-F) or Whitney U test (A, B, C, G).

✱✱✱Figure 5 .
Figure 5. Compromised IL-17A response in TLR2-deficient Vγ6 cells is due to reduced expression of IκBζ TLR2 -/-or WT mice were associated with C. mast on their ocular surface.(A) RNA-seq was performed on Vγ6 T cells isolated from the indicated mice.The PCA plot shows 4 groups: cells from naïve WT or TLR2 -/-mice, and cells from C. mast + WT or -/-mice.N= 3 per group.(B) Z scores of selected IL-17 pathway-related genes in WT and TLR2 -/-Vγ6 cells from C. mast + mice.Blue bars represent genes downregulated in TLR2 -/-Vγ6 cells.Z scores = (mean gene counts in TLR2 -/-C.mast + group -mean gene counts in WT C. mast + group)/ SD of corresponding WT C. mast+ group's counts.(C) ATAC-seq data showing traces, to the same scale, for Il17a genomic region of CD27-CD44 high Vγ6 from C. mast + WT and TLR2 -/-mice.N= 2 per group.(D) Bar plots show the reads of nfkbiz (encodes IκBζ) from RNA-seq of Vγ6 cells.N= 3 per group.Vγ1 -Vγ4 - cells(Vγ6 )were sorted from WT and TLR2 -/-mice after C .mast inoculation.The gene expression of nfkbiz in sorted Vγ6 cells was quantified by real-time PCR and normalized to β-actin.N= 4-6 per group.(E) Vγ6 cells were stimulated with ΙL-7 alone (Control) or IL-7 plus TLR2 ligand Pam3CSK4 for 2 days.The expression of Nfkbiz and Il17a was quantified by real-time PCR and normalized to β-actin.Data were combined from 2 experiments.N=4.(F) Vγ6 cells were transfected with pooled CRISPR ribonucleoprotein complexes, targeting different regions of Nfkbiz (sg1 and sg2) or a non-targeting control (NTC) that does not target the mouse reference genomes.Three days later, CRISPR-edited samples stimulated with PAM3CSK4 for one day were evaluated for Il-17a expression by real-time PCR.Data were combined from 2 experiments.N=6.Bars represent mean ± SEM.Significance was determined by Welch's t-test (D-F).

Figure 7 .
Figure 7. Impaired Cpt1 function underlines the defective IL-17A response of TLR2-deficient Vγ6 T cells and is also made available for use under a CC0 license.wasnot certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105