Dectin-1 stimulation promotes a distinct inflammatory signature in the setting of HIV-infection and aging

Introduction

Dectin-1(CLEC7A) is a member of the C-type lectin receptor family that recognizes carbohydrate structures on pathogens, with Dectin-1 specifically recognizing and binding β-1,3/1,6--glucans [1–3], a component of the fungal cell wall. Dectin-1, originally identified as “dendritic-cell-associated C-type lectin-1” [4] is primarily expressed on myeloid cells, including dendritic cells, monocytes, macrophages, and neutrophils. Dectin-1 expression has also been noted on non-immune cells such as lung epithelium [5] and intestinal M cells [6]. Through the recognition of β-glucans, Dectin-1 can recognize human pathogens that are particularly important causes for infection in immunocompromised hosts, such as Aspergillus, Candida, Coccidioides, Talaromyces, and Pneumocystis [1, 2]. Binding and stimulation of Dectin-1 leads to phosphorylation of its ITAM motif, subsequent downstream signaling through the Syk/CARD9 pathway that ultimately results in NF-κB activation and inflammatory cytokine production. Dectin-1 activation also induces activation of the NLRP3 inflammasome and a Th-17 response [1]—playing a major role in anti-fungal immunity. Additionally, Dectin-1 may also recognize other microbial pathogens such as bacteria (Salmonella), mycobacteria [7, 8] as well as endogenous ligands [9] that may contribute to age-related chronic inflammation.

Invasive fungal infections in humans carry unacceptably high rates of mortality that can exceed 50% despite anti-fungal therapies. Patients with HIV-infection [10], and increased age [11, 12] are at high risk but the effects of HIV-infection and age on Dectin-1 function remain unknown. Consequently, we evaluated Dectin-1 function in a cohort of HIV-infected and uninfected young and older adults. Peripheral mononuclear cells (PBMCs) were stimulated with whole glucan particles (1,3/1,6-β-glucan from S. cerevisiae) which specifically stimulate Dectin-1 and lack TLR stimulating activity. Using multicolor flow cytometry, we evaluated monocytes, the most abundant innate cells in the peripheral blood functioning as antigen presenting cells that may differentiate into macrophages and dendritic cells at sites of inflammation. Monocytes can be subdivided into the following subsets as defined by their expression of CD14 and CD16: classical (CD14+CD16lo), inflammatory (CD14+CD16+, known to be expanded in the setting of aging and HIV infection) [13], and non-classical (CD14lo CD16hi) subset (also increased with HIV disease). Monocytes can also be further defined by the expression of activation markers such as CD11b of which the expression is also increased in the setting of aging and HIV infection [13–17]. Dectin-1 function was also studied in human dendritic cells which are potent antigen presenting cells found in the peripheral blood and lymphoid tissue. Dendritic cells can be subdivided into myeloid dendritic cells (CD11c+, CD123-; mDCs), and plasmacytoid dendritic cells (CD11c-, CD123+; pDCs) via expression of CD11c and CD123 [18] as previously described. Finally, we performed RNA-seq analysis on sorted inflammatory monocytes to elucidate the effects of age and HIV infection on transcriptomic signatures at baseline and upon Dectin-1 activation.

Results

We recruited a total of 81 HIV-negative and HIV-positive participants, divided between younger and older adults (as noted in the Methods); the characteristics of enrolled individuals are shown in Table 1. The HIV-positive and HIV-negative groups were comparable in age and gender distribution, incidence of comorbidities such as diabetes, metabolic syndrome, cardiovascular disease and pulmonary disease. HIV-positive adults, compared to the HIV-negative cohort, had higher rates of recreational drug use, number of co-morbidities and differed in distribution of self-reported race and Hispanic ethnicity. Nearly all of the HIV-infected cohort were on antiretroviral therapy (ART) and had CD4 counts >200/mm³. However, to account for the effects of differing years with HIV-infection, percent lifespan with HIV-infection (since diagnosis) was calculated for each HIV-infected subject and is represented as a median with an interquartile range (25 percentile, 75 percentile). Of note, there were four congenitally HIV-infected subjects, all in the young group. Please note that these participants are further stratified by age and HIV status in Supplementary Table 3.

Freshly isolated PBMCs were stimulated with whole glucan particles (1,3/1,6-β-glucan, WGP Dispersible), a specific Dectin-1 agonist [19] and analyzed via intracellular cytokine staining (ICS) and multicolor flow cytometry. A soluble form of WGP that binds to the Dectin-1 receptor without activation was used as a negative control as noted in the Methods. The soluble form of WGP did not elicit any cytokine production (Supplementary Figure 14). We determined intracellular IL-10, IL-12, IL-6 and TNF-α production in classical (CD14+CD16lo), inflammatory (CD14+CD16+), activated (CD11b+CD14+) and non-classical monocytes (CD14loCD16H) monocytes [15]. In activated CD11b+ CD14+ monocytes, we found an age-associated increase in Dectin-1-induced IL-6, IL-12 and TNF-α production in older, compared to young, HIV-negative adults. Furthermore, TNF-α and IL-12 production in these activated monocytes was increased in both young and older adults with HIV infection, when compared to HIV-negative adults, respectively (Figure 1A). Robust IL-10 production appeared comparable in all cohorts. These results in CD11b+ monocytes reflect an age- and HIV-associated heightened activation of Dectin-1 function. CD14+ CD16+ inflammatory monocytes also showed an HIV-associated increase in TNF-α production (HIV-positive vs. HIV negative individuals) in both young and older adults. An HIV-associated increase in IL-12 levels was also noted in HIV-positive young, when compared to HIV-negative young individuals (Figure 1B). Classical monocytes (CD14+CD16lo) demonstrated an age-associated increase in production of IL-12 and IL-6, but in contrast to CD14+ CD11b+ or inflammatory monocytes a statistically significant effect of HIV infection was not found (Supplementary Figure 2A). In contrast, non-classical CD14+CD16H monocytes from HIV-negative adults showed a significant age-associated decrease in intracellular IL-12 levels, with a trend of decreased TNF-α production that did not reach significance. Cytokine production of IL-12 and TNF-α in HIV-positive young and older adults was comparable to the young HIV-negative cohort (Supplementary Figure 2A, 2B). Taken together, these results indicate that activated CD11b+ and inflammatory monocytes show a HIV-associated increase in TNF-α and IL-12 production. In the CD11b+ activated subset, we also observed an age associated increase in IL-12, TNF-α and IL-6 production. Baseline cytokine data shown in Supplementary Figure 15, show increased basal levels of TNF-α, IL-12, IL-6, and IL-10 in the CD14+CD16+ in the setting of HIV-infection. This trend is also noted in the CD11b+CD14+ with the exception of TNF-α which showed decreased levels in the setting of HIV-infection. General trends of cell percentages for monocytes before and after stimulation are shown in Supplementary Figure 13.

To study the basis for altered Dectin-1-dependent cytokine production, we evaluated the effects of age and HIV infection on Dectin-1 surface expression in monocyte subsets (Figure 2). We found significantly increased surface expression of Dectin-1 in both older adults and HIV-infected individuals in all monocyte populations with the exception of the nonclassical (CD14+ CD16H) where we only saw an HIV-associated difference in Dectin-1 surface expression. For HIV-positive young adults, Dectin-1 expression was increased in all monocyte subsets compared to HIV-negative young individuals, and we found a similar increase in HIV-positive older, compared to HIV-negative older adults that was statistically significant for the CD14+ CD11b+ and CD14+ CD16H subsets. For the activated CD14+ CD11b+ monocytes, in which we saw both an age and HIV-associated increase in Dectin-1 surface expression, we built a multivariable regression model to incorporate the effects of clinical covariates (Table 2). In the unadjusted model, both age and HIV-infection were significant and non-interacting contributors. However, the addition to the model the co-variates of recreational drug use, history of fungal disease, number of medical co-morbidities, and percent lifespan with HIV led to a loss of significance for age. In the fully adjusted model, Dectin-1 surface expression was significantly associated with HIV-infection, percent lifespan with HIV, recreational drug use, and number of co-morbidities. When IL-12 in CD14+CD11b+ activated monocytes was evaluated in a multivariable regression model (Supplementary Table 4) both age and HIV-infection remained significant contributors even when adjusted for drug use, fungal disease, co-morbid conditions, and % lifespan with HIV. Only age remained significant when TNF-α was evaluated in activated monocytes within an adjusted model.

We also examined the response of Dectin-1 stimulation in dendritic cells (DCs) in the context of aging and HIV-infection. Lineage negative and HLA-DR positive cells were separated into CD11c+, CD123- (mDCs), and CD11c-, CD123+ (pDCs) [18]. We determined intracellular production of IFN-α, TNF-α, IL-6 and IL-12 in both subsets following WGP stimulation. mDCs (Figure 1C) showed a significant age-associated increase in IFN-α and TNF-α production with Dectin-1 stimulation, and an HIV-associated increase in IFN-α and IL-12 production in both young and older adults. IL-6 production was comparable among these groups except for a significant increase in HIV-positive versus HIV-negative older adults. Cytokine production in pDCs (Figure 1D) was comparable in HIV-negative young and older adults. However, there was a significant HIV-associated increase in IFN-α, TNF-α (in HIV-positive young) and IL-6 and IL-12 (in HIV-positive older) production. Dectin-1 surface expression levels in mDCs and pDCs showed a significant HIV-associated increase when compared to HIV-negative adults (Figure 2E). Baseline cytokine data shown in Supplementary Figure 16 show an increase in basal levels of TNF-α and IL-12 in mDCs in the HIV-infected cohorts. IL-12 basal levels were also noted to be increased with HIV-infection in the pDC population. Basal IL-6 levels interestingly were increased in the young HIV-negative cohort compared to other groups. Basal IFN-α were comparable between all groups in both mDCs and pDCs. Overall, our findings show that Dectin-1-dependent type I interferon production is increased with HIV infection in mDCs and pDCs from both young and older adults, with age-related increases in IFN-α and TNF-α found in mDCS from HIV-negative older adults.

Discussion

To our knowledge, our study is the first to evaluate the specific function of Dectin-1 in the setting of aging and HIV-infection. A few themes emerged from our studies. Stimulation of both monocytes and dendritic cells with β-D-glucans led to a more pro-inflammatory phenotype in both monocytes and dendritic cells in HIV-infected individuals that included increased levels of IL-12, TNF-α, and IFN-α. While this pro-inflammatory phenotype was evident in certain cell types (CD11b+ activated monocytes, myeloid dendritic cells) for HIV-negative older adults, the most striking differences were associated with HIV-infection. Our previous studies of Mincle, another C-type lectin receptor family member recognizing carbohydrate motifs including M. tuberculosis cord factor, also showed an increase in cytokine production in monocytes with age and HIV infection [32]; thus HIV and age appear to enhance cytokine production downstream of C-type lectin signaling and represent an additional factor that could contribute to age-related chronic inflammation.

Our analyses of RNA-seq experiments on purified CD14+ CD16+ monocytes from young and older adults with and without HIV infection were notable for a distinct inflammatory signature in the HIV-older population. Gene expression from several pro-inflammatory pathways was enhanced at baseline in this group, such as TNF-α signaling (and in this regard, Dectin-1-dependent TNF-α production in inflammatory monocytes was increased with HIV infection in both young and older adults (Figure 1)). Increased GSEA enrichment of the IL-2/STAT5 signaling pathways was also noted in the HIV-positive Older cohorts (also seen in HIV-negative Older cohort). The IL-2/STAT5 signaling pathway although traditionally thought to be a T cell pathway, has been reported in monocytes where it may play a role in anti-fungal immunity [33]. Additionally, there was also increased baseline expression of IL-19/IL-24 (Supplementary Figure 4A), and the KEGG complement/coagulation pathways (Supplementary Figure 4C). The link between Dectin-1 and coagulation factors is unclear, but both F3 (tissue factor and PLAUR (plasminogen activator) were hub genes only for the HIV-Older group (Figure 5A). Because increased production of clotting factors is also associated with age-associated chronic inflammation [13], our results suggest CLR signaling as an additional contributing factor. Finally, increased Reactive Oxygen species (ROS) pathway signaling was noted in the Older HIV-negative and HIV-positive cohorts (with stimulation) (Figure 4). However, baseline analysis only showed increased enrichment of the ROS signaling in the HIV-Older cohort (Supplementary Figure 7). Overall, HIV-infected older adults demonstrated unique gene expression with Dectin-1 stimulation when compared to the other cohorts.

Studies in the early years of the HIV pandemic reported higher plasma levels of IFN-α in HIV-infected individuals in both acute and chronic infection [34] that was strongly associated with the immune activation seen with chronic HIV-infection and is thought to contribute to pathogenesis. Our studies indicate that Dectin-1 signaling, particularly in HIV-infected individuals, may contribute to this dysregulated IFN production. Notably, both mDCs and pDCs showed an HIV-associated increase in Dectin-1-induced IFN-α production in young and older adults (Figure 1C, 1D). This increase in IFN-α production also paralleled the increased Dectin-1 surface expression in both mDCs and pDCs noted with HIV-infection (Figure 2E). We also found increased enrichment of Dectin-1-dependent IFN-α and IFN-γ pathway expression only in the HIV-Older group, in contrast with downregulated IFNα and IFN-γ responses in all other cohorts (Figure 4 and Supplementary Figure 5). In this regard, we found an age-associated decrease in basal IFN-α and IFN-γ pathway expression that may contribute to enhanced Dectin-1-dependent IFN signaling in HIV-Older adults. A post-hoc analysis of monocytes (done after RNA-seq results) did demonstrate that both IFN-α and IFN-γ are produced in monocytes in response to Dectin-1 stimulation (Supplementary Figure 11). Interestingly, we found both IFN-α and IFN-γ levels at baseline to be increased in the HIV-older cohort when compared to HIV-negative young and older adults. Dectin-1 stimulation showed comparable levels of IFN-α production amongst the cohorts, with a significant difference in IFN-γ production where HIV-older adults demonstrated decreased IFN-γ production when compared to HIV-negative young adults. Although controversial, IFN-γ production has been definitely noted in antigen presenting cells (myeloid cells such as monocytes) in prior studies [35]. IFN-α has been reported to downregulate the production of IFN-γ by antigen presenting cells [36]—in this case the strong IFN-α signature seen in the HIV-older cohort could contribute to the decreased IFN-γ production seen in HIV-older adults in response to Dectin-1 stimulation. However, the already elevated levels of IFN-γ seen at baseline in HIV-older adults points to dysregulation of the IFN pathways in the setting of HIV-aging. While traditionally associated with antiviral immunity, there is accumulating evidence that interferon responses (Type I and Type III) also modulate the antifungal immune response [37–39]. Production of IFN-γ has not been previously reported in myeloid cells in response to Dectin-1 stimulation—although, treating myeloid cells with IFN-γ prior to a fungal challenge was associated with enhanced inflammation [40, 41]. Although, type I IFN production may play an important role in the antifungal response, it may also result in dysregulated inflammatory responses in monocytes that lead to worsened outcomes from fungal infection [42] as can be seen in older adults with HIV infection. Additionally, the increased and persistent production of IFNs via Dectin-1 stimulation may also be contributing to the pro-inflammatory environment that is seen in HIV-infection and Aging.

Analysis of our RNA-seq data revealed the innate immune receptor Triggering receptor in myeloid cells-1 (TREM-1) as a significant upstream regulator in all cohorts (Figure 5B). There are limited studies demonstrating a relationship between TREM-1 and Dectin-1 in fungal pathogenesis, although expression of TREM-1 and Dectin-1 both increased with severity and progression of an Aspergillus keratitis mouse model [21], with inhibition of both TREM-1 and Dectin-1 resulting in protection from fungal disease. TREM-1 is a receptor expressed on neutrophils and monocytes that modulates the inflammatory response [43, 44], and is upregulated in sepsis. Although, the specific agonist for TREM-1 remains unknown, the receptor is known to interact with CARD9 in a complex with Bcl-10 and MALT-1, hinting at a possible interaction with C-type lectin receptors [43]. The significant upregulation of TREM-1 noted in our study also alludes to a physical interaction between TREM-1 and Dectin-1.

Other upstream regulators that emerged included the cytokines TNF-α, IL-1β that were significant in all cohorts, and IFNs that were prominent negative regulators in the HIV-Young cohort. Other important regulators that emerged was the NF-κB complex, and the MAP kinases ERK, and p38 pathways which have been reported as important components of the Dectin-1 signaling [1, 45]. In fact, ERK and p38 were shown to regulate zymosan induced IL-10 production in a mouse model [45]. IL-10 was robustly produced by all cohorts in our study in response to Dectin-1 stimulation. Dectin-1 stimulation also increased expression of the anti-inflammatory cytokines IL-19 and IL-24 in the CD14+CD16+ monocytes in most cohorts, with the HIV-Older cohort already increased at baseline (Supplementary Figure 4A).

Other notable differences should be pointed out. The HIV-Older group showed decreased gene expression of IL-7R gene expression with Dectin-1 stimulation when compared to all other cohorts (Supplementary Figure 4A). Expression of IL-7R in monocytes has been reported in the setting of LPS exposure and its role remains to be fully defined [46]. Decreased expression of IL-7R in monocytes has not been reported in the setting of HIV and aging. However, decreases in IL-7R expression and impaired IL-7 signaling in T cells have been reported in the setting of both aging [47] and HIV-infection [48]. Another gene of note was IL-1β which was noted to be increased at baseline in all cohorts except for the HIV-Older cohort (Supplementary Figure 4A). Dectin-1 stimulation led to an upregulation of IL1-β, in all cohorts (Supplementary Figure 4A–4D), although higher in all groups compared to the HIV Older group. Dectin-1 is a known inducer of the inflammasome [49, 50]. Interestingly, IL-1β emerged as a significant upstream regulator of Dectin-1 signaling in all cohorts, in addition to an important hub gene (most cohorts) as noted in Figure 5A, 5B. Further study of the Dectin-1 induced inflammasome is needed in the setting of HIV-infection and aging.

Overall, Dectin-1 stimulation led to the upregulation of several metabolic pathways, including mTORC1 signaling, the hypoxia pathway, and P13K-Akt signaling. Upregulation of the MTORC1 signaling and the Hypoxia pathway was noted all cohorts (Figure 4). Baseline GSEA pathway analysis also demonstrated increased MTORC1 pathway enrichment in HIV-Older adults which was absent in all other cohorts. (Supplementary Figure 7). KEGG gene enrichment analysis showed upregulation of PI3K-Akt pathway in all groups except the HIV Old cohort (Supplementary Figure 3), although was also noted to be upregulated at baseline (Supplementary Figure 6). Activation of the Akt/mTOR/HIFβ–α pathway is thought to be the metabolic basis for trained immunity in Monocytes trained with β–glucan [51], and trained immunity is likely playing a role in the anti-fungal response in humans. Our findings show that trained immunity may also be contributing to the sustained pro-inflammatory phenotype seen in the setting of HIV and aging, a finding which has also been noted in other studies [52]. Furthermore, Dectin-1 stimulation also led to a strong upregulation of the unfolded protein response in all cohorts (Figure 4). There is evidence that the unfolded protein response may be important for proper innate immune function and may be necessary for the proper functioning of dendritic cells [53].

Finally, Dectin-1 led to the upregulation of several differentiation signatures (Figure 6) alluding to the possibility that Dectin-1 stimulation may lead to the differentiation of monocytes into both dendritic cells and macrophages. Specifically, Dectin-1 induced transcriptomic changes in monocytes suggest the differentiation of CD14+CD16+ monocytes mainly to monocyte-derived dendritic cells—changes that are noted in all the cohorts (Figure 6B). However, the transcriptomic profile of the stimulated inflammatory monocytes of HIV-positive older adults also revealed a distinct gene signature that demonstrated an activated DC signature, as well as a more inflammatory M1 macrophage signature. A prominent activated DC signature was also seen in Older HIV-negative adults with co-morbidities (a smaller upregulation was noted in Young HIV-negative adults with co-morbidities). Young HIV-negative adults seemed to skew towards a non-activated dendritic cell signature. Activation of the PPAR-γ pathway has been shown to inhibit Dectin-1 induced dendritic cell activation in human PBMCs [54] from healthy volunteers. Interestingly, KEGG DEG analysis did not show upregulation of the PPAR signaling pathway in the HIV-Older and Older HIV-negative group as compared to young adults where it was upregulated. However, baseline pathway analysis also showed upregulation of PPAR signaling at baseline in the HIV Older cohort—alluding to more complex signaling in the setting of HIV and aging. Overall, Dectin-1 signaling in the setting of HIV and aging was associated with a more inflammatory activated DC signature and M1 macrophage signature. Additionally, the output of inflammatory mediators brought on by Dectin-1 simulation could promote different T cell responses. In fact, Dectin-1 stimulation in dendritic cells has been shown to promote Th17, Th1, Th2 and cytotoxic CD8 responses [55]. Future experiments will be needed to discern how HIV and aging affect these downstream T cell responses from Dectin-1 stimulation.

Other roles for Dectin-1 have emerged which may also be playing a role in the setting of HIV-infection and aging. There is accumulating evidence that Dectin-1 recognizes endogenous ligands such as annexins, and vimentins from dying cells [9]. Vimentin when bound to Dectin-1 in atherosclerotic plaques induces ROS production in human monocytes [56]. Dectin-1 activation induced by vimentin promoted obesity and insulin resistance in a diet-induced obesity mouse model. This same study showed increased Dectin-1 expression in human adipose tissue samples of obese individuals when compared to lean subjects [57]. Interestingly, a recent study of caloric restriction in humans showed a downregulation of Dectin-1 signaling in RNA seq studies of subcutaneous adipose tissue after two years of 14% caloric restriction [58]. In our study, Dectin-1 surface expression in activated monocytes was examined in the context of a multivariable regression model (Table 2). Within our fully adjusted model, the co-variates of HIV-infection, recreational drug use, number of co-morbidities, and percent lifespan with HIV all proved to be significant. The significance of number of co-morbidities in the model of Dectin-1 surface expression suggests that factors unrelated to fungi are important for Dectin-1 surface expression and need to be further explored. Therefore, the stimulation of Dectin-1 by endogenous ligands could certainly be contributing to both age and HIV-associated chronic inflammation as seen in our study.

In addition to endogenous ligands, circulating fungal ligands could also be contributing to these notable age and HIV-associated differences. Elevated levels of circulating β-D-glucan levels have been noted in several studies in the setting of chronic HIV-infection that persist despite long term Antiretroviral therapy (ART) and is thought to be secondary to microbial translocation [59, 60]. Previous studies have shown that elevated levels of β-D-glucan was associated with markers of immune activation (soluble CD14, HLA-DR+ CD4/CD8 T cells, and IL-6/IL-8 levels) and decreased Dectin-1 surface expression on monocytes from individuals with HIV infection, compared to HIV-negative controls [59]. HIV-positive and negative individuals in this study were not segregated into age groups, and so it is not clear if the general HIV-associated increase in Dectin-1 expression we observed would have been found; in addition, there were methodological differences between our studies, such as the analysis of freshly isolated PBMCs (this study), versus cryopreserved samples. Our findings showed increased Dectin-1 surface protein expression with both increased age and HIV-infection. Evaluation of Dectin-1 (Clec7a expression) gene expression (Supplementary Figure 10) showed an opposite trend of decreased gene expression levels in the HIV-Older cohort and increased levels in the young HIV-negative cohort. These findings suggest a mechanism that is regulated post-transcriptionally in the HIV-Older cohort that needs to be further investigated. A recent paper that examined monocytes from people living with HIV found a strong correlation between serum levels of β-D-glucans and production of IL-1β in general (although specific stimulation with Candida albicans led to decreased cytokine production in this study) and suggested that the robust production of cytokines by monocytes in HIV-infection was linked to trained immunity in monocytes [52]. Microbial translocation is something that is also likely occurring with increased age [61] as increased levels of surrogate markers of microbial translocation have been noted in older adults. Therefore, our findings of a unique pro-inflammatory signature in the setting of HIV and aging could be secondary to elevated levels of circulating of β-D-glucans that have been noted in the setting of chronic HIV-infection [13].

Overall, this study demonstrates that age, HIV-infection and co-morbidities can alter the individual immune response. In particular our study showed a unique immune signature in the setting of both HIV and aging in response to Dectin-1 stimulation. As our HIV-positive population ages these immune differences will need to be considered for future vaccines and therapeutics. Dectin-1 agonists such as β-D-Glucans have been proposed to be possible adjuvants for vaccines as they induce epigenetic changes associated with trained immunity [62, 63]—and so could represent a useful immune response. However, Dectin-1 induced inflammation possibly mediated by trained immunity could also contribute to the immune activation or chronic inflammation that is seen in the HIV-infected population. Therefore, Dectin-1 induced inflammation could represent a double-edged sword in the HIV/Aging population. Further studies are needed to dissect these mechanisms. At the very least we need to consider the HIV/Aging population as a unique population in clinical studies given their unique inflammatory profile.

Materials and Methods

Supplementary Materials

Supplementary Figures

Supplementary Table 1

Supplementary Tables 2-4

Supplementary Table 5