Identification of a reactive astrocyte subpopulation during HIV-associated pain pathogenesis in mouse models

Domain: biology
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Pain is a common complication in individuals with HIV, yet the underlying pathogenic mechanisms remain poorly understood. Reactive astrocytes are selectively activated in the spinal dorsal horn of HIV patients experiencing pathological pain, implicating their potential role in pain pathogenesis. However, the specific pathogenic subtype of these reactive astrocytes has not been defined. Here, we identify a distinct subpopulation of pain-associated reactive astrocytes in the lumbar spinal cord of HIV-1 gp120 transgenic mice using single-nucleus RNA sequencing. This astrocyte subpopulation displays transcriptomic features associated with phagocytosis and inflammation and depends on galectin-3 for its formation. Both global and astrocyte-specific heterozygous knockout of Lgals3 (galectin-3) markedly reduce mechanical allodynia in gp120 transgenic mice. These findings reveal a subset of reactive astrocytes that play a role in remodeling the spinal pain circuitry underlying HIV-associated pain.

Introduction
Pathological pain is a frequent comorbidity in HIV patients, but its underlying mechanism remains unclear1,2. Among the neuropathologies observed in the pain neural circuits of HIV patients, reactive astrocytes in the spinal cord dorsal horn (SDH) are particularly evident3. Importantly, reactive astrocytes were found in the SDH of HIV patients who developed neuropathic pain but not in those without pain disorders3. These findings on HIV patient spinal specimens suggest that reactive astrocytes play a crucial role in the pathogenesis of HIV-associated pain.

Under normal conditions, spinal astrocytes maintain circuitry homeostasis by modulating pain processing in neuronal circuits via their various biological activities, such as neurotransmitter uptake and gliotransmitter release4. When the homeostatic function is disrupted in reactive astrocytes, their associated pain neuronal circuits may become sensitized to activation. Reactive astrocytes in the SDH have been reported in animal models of various pathological pain types4,5, including HIV-associated pain models6. Supporting their critical role in pain pathogenesis, reactive astrocyte ablation blocks mechanical allodynia induced by HIV-1 glycoprotein 120 (gp120)7. Concomitantly, astrocytic ablation also reverses gp120-induced neural circuit polarization (NCP) implicated in HIV-associated pain pathogenesis7. Similarly, ablation of reactive astrocytes blocks opioid-induced hyperalgesia and its associated NCP in the SDH8.

Astrocytes are heterogenous in function, morphology, and spatio-temporal distribution across CNS regions9,10. Astrocytes undergo a reactive transformation in various disease conditions. Recent data from single-cell RNA sequencing studies found reactive astrocytes can give rise to a spectrum of subtypes with diverse functions11. These astrocyte subtypes have characteristic gene expression signatures implicated in specific cellular pathways, such as extracellular matrix remodeling12, plaque degradation13, inflammation, and anti-inflammation14, and may contribute to relevant pathogenesis of specific diseases. However, despite the emerging consensus about the critical role of reactive astrocytes in pain pathogenesis4,5, little is known about pain-pathogenic subtypes of astrocytes and their contribution to underlying pathogenesis.

In this study, we performed snRNA-seq analysis of the lumbar spinal cord of HIV-1 gp120 transgenic (gp120Tg) mice15, a model of HIV-associated pain, and identified a subpopulation of reactive astrocytes, named HIV pain-associated astrocytes (HIPAs). This astrocytic population has transcriptomic signatures of phagocytosis and inflammation and is a phagocyte of neurons. In addition, global knockout (KO) of Gal3 lead to depletion of this astrocytic population and blockage of the development of mechanical and thermal allodynia in gp120Tg mice. Conditional KO of one copy of Gal3 in astrocytes also attenuated mechanical allodynia. These findings suggest that HIPAs play an important role in the pathogenesis of HIV-associated pain.

Results
Identification of an astrocytic subpopulation specific to the spinal cord of gp120 transgenic mice by snRNA-seq analysis
To investigate the cellular mechanism underlying pain pathogenesis induced by HIV-1 gp120, we performed snRNA-seq analysis of spinal cords from Gfap-gp120 transgenic (gp120Tg) mice at 4–5 months of age. Single nuclei were extracted from the lumbar regions of the cord and subjected to cDNA synthesis using 10X Genomics (10X Chromium v3 chemistry). Barcoded cDNA libraries were sequenced using next-generation sequencing, and the snRNA-seq data were processed and analyzed for quality control using Seruat16. Using UMAP, we performed clustering analysis of snRNA-seq data from 20,284 nuclei that passed the quality control filter. The data was derived from wild-type (WT) mice (a pool of spinal lumbar sections from three mice) and gp120Tg mice (a pool of lumbar sections from three mice). This analysis generated a detailed atlas of transcriptomically distinct spinal cell clusters, including various types of neurons and glia (Fig. 1a).

Previous studies have suggested an association of reactive astrocytes in the spinal dorsal horn (SDH) with the development of neuropathic pain in HIV patients3. Using a gp120 model mouse, we confirmed the role of reactive astrocytes in the pathogenesis of HIV-associated pain7. To identify gp120-induced specific subtypes of astrocytes contributing to pain pathogenesis, we subset astrocytic clusters revealed by feature plots of marker genes Aqp4 and Gja1 (Fig. 1b, c). These pooled astrocytes were subjected to further dimension reduction analysis17. From this analysis, we identified eight astrocytic subclusters from the combined WT and gp120Tg astrocytes (Fig. 1d). Splitting the clusters based on their genotypes (WT vs. gp120Tg) revealed marked differences between WT and gp120Tg astrocytic clusters (Fig. 1e, f). Supplementary Data 1 lists the number of astrocytes in each cluster in the WT and gp120Tg mice. Compared to their WT counterparts, the gp120Tg astrocytes in clusters 1 and 2 were significantly decreased, while those in cluster 5 were increased. Cluster 7 was specifically identified in gp120Tg mice (Fig. 1e–g).

To gain insights into the biological functions of individual astrocytic clusters in the WT and gp120Tg datasets, we used Seurat FindAllMarkers to identify specific marker genes for each cluster in the WT and gp120Tg mice (Supplementary Data 2; Supplementary Data 3). These markers represent genes that are significantly upregulated or downregulated (adjusted p-value<0.05) in a given astrocytic cluster compared to all other astrocytic clusters. As shown in Fig. 1h, the top 10 cluster-specific marker genes in the WT dataset exemplified the expression of the different clusters. We found that the WT astrocytic clusters could be separated into two major categories based on their Gfap expression levels, with clusters 1, 2, 5, and 6 showing low Gfap expression (Gfap-low) and clusters 3 and 4 showing high Gfap expression (Gfap-high) (Fig. 1h; Supplementary Fig. 1a), consistent with previous observations13. Clusters 1 and 2 were the two major subpopulations expressing Slc6a11 and Slc7a10 (Fig. 1h). Slc6a11 encodes an astrocyte neurotransmitter transporter that plays a key role in GABA uptake18, while Slc7a10 encodes a sodium-independent amino acid transporter with a primary role related to modulation of excitatory glutamatergic neurotransmission19. Clusters 3 and 4 expressed high Gfap levels but low levels of Slc6a11 and Slc7a10 (Fig. 1h).

In the gp120Tg mice, there was a clear shift from Gfap-low to Gfap-high populations in the astrocytic clusters (Supplementary Fig. 1b). To understand how the WT astrocytic clusters transformed into the gp120Tg astrocytic clusters, we examined the expression of the top 10 WT cluster-specific marker genes in the gp120Tg astrocytes (Fig. 1i). In the gp120Tg mice, there are eight clusters. Since clusters 6 and 8 are very small subpopulations, we did not pursue them in this study. From the gp120Tg heatmap (Fig. 1i), we can see that the marker genes of WT clusters 1 and 2 are highly expressed in gp120Tg clusters 1, 2, and 5. The WT marker genes of clusters 3 and 4 are highly expressed in gp120Tg clusters 3 and 4. These data suggest that, in gp120Tg mice, clusters 1, 2, and 5 originated from WT clusters 1 and 2, whereas clusters 3 and 4 were derived from WT clusters 3 and 4. Notably, cluster 7 did not express any WT cluster-specific marker genes, suggesting that cluster 7 is a de novo astrocyte cluster formed only in gp120Tg mice.

To further determine the transcriptomic signature of Cluster 7, we generated gp120Tg cluster-specific marker gene lists (Supplementary Data 3). Figure 1j is a heatmap demonstrating the expression of the top 10 upregulated cluster-specific marker genes in gp120Tg astrocytes. The results clearly show that Cluster 7 expresses a unique set of genes. We named this cluster HIV-pain associated astrocytes (HIPA) based on their potential pathogenic contribution to pain associated with HIV infection (see below). HIPAs comprised 20% of total gp120Tg spinal astrocytes. To gain insight into their potential biological function, we imported the 112 HIPA (cluster 7)-specific marker genes from the gp120Tg dataset (Supplementary Data 3, cluster 7) into Ingenuity Pathway Analysis (IPA). IPA identified 33 well-characterized inflammatory genes (Supplementary Fig. 2) and 7 well-characterized phagocytic genes (Supplementary Fig. 3). Hence, HIPAs have enriched expression of genes related to inflammation and phagocytosis.

To visualize the expression profiles of the top IPA-selected genes related to astrocytic homeostasis, inflammation, and phagocytosis, we generated a dot plot using the combined WT and gp120Tg dataset (Fig. 1k). The plot showed that HIPAs (cluster 7) expressed low levels of homeostatic genes (Gja1, Aqp4, Slc1a2, etc.) but high levels of inflammatory genes (Gfap, Anxa3, Nox4, Cdh13, Aldh1a1, Bcl2, Adamts5, Adra1a, Col4a5) and phagocytic genes (Lgals3, Palld, Ptprj, Rsas2, C3) (Fig. 1k). These data indicate that HIPAs are both inflammatory cells and phagocytes, which may contribute to the development of pain associated with HIV infection.

Gal3 is a specific marker of HIPAs
Among the up-regulated genes related to phagocytosis or inflammation, the expression of gene coding for Gal3 (Lgals3) was high and selective in HIPAs (cluster 7) (Fig. 1k). Hence, we used Gal3 as a transcriptomic marker for HIPAs. snRNA-seq data revealed that Gal3+ astrocytes compose ~6% of the entire spinal cord astrocytes (Supplementary Data 4) and predominantly located in cluster 7 (HIPA). Gal3 was reported to play a role in neuropathic pain in a peripheral nerve injury model20,21, suggesting that HIPAs are a potentially important contributor to HIV-associated pain pathogenesis. To further validate Gal3 as a marker for HIPAs, we performed double fluorescent immunostaining of GFAP and Gal3, followed by confocal imaging (Fig. 2a, d, e). As Gal3 was reported to express in microglia in the brain22, we performed a control experiment using Iba1, a microglial marker (Fig. 2b, d). We found that Gal3 was almost exclusively expressed in astrocytes but not in microglia in the gp120Tg spinal cords (Fig. 2a, b, d, e). Approximately 95% of the Gal3+ cells were GFAP+, while ~5% of GFAP+ cells were Gal3+ microglia (Fig. 2c), indicating Gal3+ cells are predominantly astrocytes. In some cases, Gal3+ cells were observed in clumps containing both GFAP+ and Iba1+ cells (Fig. 2e). However, optical sectioning image analysis showed that Gal3 signals originated from astrocytes rather than microglia (Fig. 2f–h). These results confirm that Gal3 is a specific marker for HIPAs in the gp120Tg spinal cords, as suggested by snRNA-seq data.

Differentiation of HIPAs in the spinal cord
To gain insights into the induction of HIPAs in gp120Tg spinal cords, we compared Gal3 protein levels in the spinal cord of WT and gp120Tg mice by western blotting (Fig. 3a) and found that Gal3 expression was low in WT but significantly upregulated in gp120Tg spinal cords (Fig. 3a). Immunostaining analysis confirmed Gal3 upregulation in the spinal cord of gp120Tg mice, with the induction of HIPAs in gp120Tg spinal cords (Fig. 3b–e). The increase of Gal3+ astrocytes in the gp120Tg spinal cord was further validated by the co-staining of Gal3 with SOX9 and GFAP (Supplementary Fig. 4). To demonstrate the Gal3 is specifically induced by gp120, we compared the effect of native and heat-denatured forms of purified recombinant gp120 spinally injected (i.t.), and observed that only native gp120, but not heat-denatured gp120, induced Gal3 expression (Supplementary Fig. 5), aligning with their activity in inducing pain6.

In WT spinal cords, Gal3+ cells were largely restricted to the central canal area (Fig. 3b), with only a few sparse Gal3+ cells in dorsal horns and marginal areas (Fig. 3b). In contrast, in gp120Tg spinal cords, Gal3+ cells markedly increased, especially in the gray matter (Fig. 3c, e). Co-staining of Gal3 with GFAP and Vimentin revealed that HIPAs appeared to form a trajectory originating from the central canal to the outer margin of the gray matter in the gp120Tg mice (Fig. 3e). Concomitantly, Gal3+ cells were diminished in the central canal region of gp120Tg mice (Fig. 3c), giving an apparent impression that Gal3+ HIPAs were derived from Gal3+ precursors migrating out from the central canal region.

In the central canal of WT mice, Gal3 signals were confined to ependymal cells lining the canal (Fig. 4a). Consistent with reported findings23, Gal3+ spinal ependymal cells expressed vimentin (Fig. 3e) but not GFAP (Fig. 4b–d). In contrast, in the gp120Tg mice, Gal3 signals were completely absent from the ependymal cell layer (Fig. 4e, h). Concomitantly, many Gal3+ cells were observed outside the ependymal cell layer (Figs. 3c, e and 4e, h, i); these expressed GFAP (Figs. 3e and 4h, i) and appeared to form a trajectory toward spinal dorsal and ventral horns (Fig. 3c, e). Unlike ependymal cells in the central canal of WT mice that were GFAP-negative (Fig. 4b, c), some spinal ependymal cells in the central canal of gp120Tg mice expressed GFAP (Fig. 4f, g), signaling their commitment to an astrocytic lineage. These data are consistent with the idea that Gal3+ cells in the central canal ependymal layer may be induced to migrate out and differentiate into HIPAs in the spinal dorsal and ventral horns in gp120Tg mice.

We observed evident morphological changes of HIPAs at different positions in relation to the central canal. HIPAs in areas adjacent to the central canal ependymal layer were relatively small with shorter processes, and Gal3 protein was mainly restricted to nuclei (Fig. 4i). In contrast, HIPAs in regions farther away from the central canal, particularly in the dorsal horn, showed an increase in morphological complexity, with larger size compared to non-HIPA astrocytes (Fig. 4j) and more processes (Fig. 4j, k). In these HIPAs, Gal3 protein was observed throughout the entire cell, including the cytoplasm and processes (Fig. 4j, k). HIPAs in the spinal dorsal horn exhibited more heterogeneous in morphology, some with relatively smaller cell bodies and a few long processes and others with larger bodies and many short processes (Fig. 4k). By using Imaris AI software to create surface of DAPI staining, we also identified HIPAs with multiple nuclei (Supplementary Fig. 6).

To assess the relevance of HIPAs in the gp120Tg model, we examined spinal autopsies from HIV patients. We found that Gal3 protein was significantly induced in the spinal cord of HIV patients compared to control patients who died from non-HIV causes (Supplementary Fig. 7a). Additionally, immunofluorescent stainings showed a drastic increase in Gal3+ astrocytes in the spinal cords of HIV patients (Supplementary Fig. 7b, c), indicating that HIPAs are also induced following HIV infection, like in the gp120Tg model.

HIPAs are phagocytes of neurons
Previous studies have shown that cells expressing Gal3, such as Gal3+ macrophages, act as phagocytes and release Gal3 as an opsonin to label target cells for phagocytosis24,25. Our discovery of upregulated phagocytosis-related genes in HIPAs (Fig. 1k) suggests that this population of Gal3+ astrocytes have the potential to function as phagocytes to clear damaged neurons induced by HIV neurotoxins, such as gp120. Given that our previous work has revealed neurodegeneration in the SDH of HIV patients with neuropathic pain6 and in a mouse model of HIV-associated pain6,26, we hypothesized that HIPAs could contribute to neurodegeneration as phagocytes. To evaluate the capability of HIPAs to phagocytose neurons, we performed double fluorescent immunostaining experiments to label NeuN (a marker of neuronal soma) and Gal3. Then we used ZEISS Airyscan confocal super-resolution microscopy and took 3D Z-stack confocal images demonstrating the interaction of HIPA with neuronal soma (Fig. 5a, Supplementary Video 1). By using Imaris AI software to create solid surfaces for NeuN and Gal3 signals, we can clearly view from different angles the neuronal soma enwrapped/engulfed by a HIPA (Fig. 5b–d).

To visualize neuronal components inside HIPAs, we performed Imaris image segmentation analysis on representative Gal3+ cells (Fig. 5e). This analysis removed NeuN signals not interacting with HIPAs, allowing us to visualize the NeuN signals inside HIPAs (Fig. 5f–k). By creating solid opaque HIPA surfaces (Fig. 5f, i), transparent HIPA surfaces (Fig. 5g, j), or by removing all HIPA components (Fig. 5h, k), we were able to reveal the neuronal components engulfed by HIPAs. These image analyses showed intact neuronal somas (Fig. 5h) or debris (Fig. 5k) inside HIPAs, demonstrating that HIPAs are astrocytic phagocytes capable of phagocytosing neuronal components in the SDH of gp120Tg mice. Moreover, we made 3D videos using Imaris AI software to further confirm HIPAs phagocytosis of neuronal somas in the gp120Tg spinal cords (Supplementary Video 1, 2). Approximately 15% of Gal3+ astrocytes in the gp120Tg spinal cord demonstrated phagocyted neuronal components (Fig. 5l).

To examine the association of HIPAs with neuronal degeneration, we performed Fluoro-Jade C (FJC) staining combined with Gal3 immunofluorescent staining. We observed a significant increase of FJC signals in the spinal cords of the gp120Tg mice compared to the WT mice (Fig. 5m, n), indicating an increase of degenerated neurons. Furthermore, we found that FJC-labeled degenerated neurons co-localized with Gal3 signals (Fig. 5m), suggesting an association of HIPAs with degenerated neurons.

Gal3 knockout diminished HIPAs in gp120Tg spinal cords
Given the proposed roles of Gal3 in regulation of cell growth and differentiation27,28, we hypothesized that Gal3 is involved in the formation of HIPAs. To test this, we generated gp120Tg mice with Gal3 KO (gp120Tg/Gal3−/−) by crossing gp120Tg mice with mice carrying a Gal3 targeting mutation29. Western blotting analysis confirmed that Gal3 KO significantly attenuated the upregulation of Vim and GFAP protein expression in the gp120Tg spinal cord (Supplementary Fig. 8a–c), indicating that Gal3 is important for the astrocytic reaction induced by gp120.

We then performed snRNA-seq analysis to investigate if HIPAs could still form in gp120Tg/Gal3−/− mice. Integrated UMAP clustering analysis of pooled 48,935 lumbar spinal nuclei from wild-type (n=3), gp120Tg (n=3), gp120Tg/Gal3−/− (n=3), and Gal3−/− (n=3) mice showed all major spinal cell types, including different types of neurons and glia (Fig. 6a). Astrocytes were subset for a second-dimension reduction analysis (Fig. 6b, c) and the astrocytic clusters were displayed in an integrated UMAP plot (Fig. 6d). Splitting the astrocytic clusters based on their genotypes revealed that, similar to the results shown in Fig. 1e, f, cluster 7 (HIPAs) was specifically observed in the gp120Tg mice and largely disappeared in WT (Fig. 6e–f). Importantly, this group of astrocytes induced in gp120Tg mice was largely absent in gp120Tg/Gal3−/− mice (Fig. 6g, i), suggesting that Gal3 KO prevented HIPA formation induced by gp120. Moreover, expression of the top 50 upregulated genes in HIPAs, including genes involved in inflammatory and phagocytic pathways, were also dramatically attenuated in the gp120Tg/Gal3−/− mice (Fig. 6j–l). Astrocytes expressing inflammatory genes (e.g., Anxa3) and phagocytotic gene (e.g., C3), which were increased in the gp120Tg mice (Fig. 6o, s) compared to WT (Fig. 6n, r) and primarily in cluster 7 (HIPAs), were diminished in the gp120Tg/Gal3−/− mice (Fig. 6p, t). In contrast, Gal3 KO in WT mice did not induce evident changes in astrocytic clusters (Fig. 6e, h, n, q, r, u) and the expression profiles of HIPA-associated genes (Fig. 6m). To confirm the effect of Gal3 KO on HIPA formation, we performed RNAscope analysis of C3, whose expression is enriched in HIPAs (Fig. 1k). The results showed that C3+ astrocytes increased in gp120Tg mice and the increase was blocked in gp120Tg/Gal3−/− mice (Supplementary Fig. 9). These data strongly suggest that Gal3 induced by gp120 is important for the formation of HIPAs.

Gal3 KO attenuates pathogenic processes of gp120-induced pain
Next, we sought to understand the potential contribution of the HIPA population of reactive astrocytes to pain pathogenesis. Firstly, we determined the effect of Gal3 KO on mechanical allodynia in gp120Tg mice. As previous studies have shown, young adult gp120Tg mice (2 months) do not manifest mechanical allodynia measured by von Frey testing30. However, we observed that mechanical allodynia began to manifest at later ages (4–5 months) (Fig. 7a). Importantly, expression of mechanical allodynia in gp120Tg mice (4-5 months) was blocked by global KO of Gal3 (gp120Tg/Gal3−/−) or heterozygous KO of astrocytic Gal3 in the gp120Tg mice (gp120Tg/Gal3CKO+/−) (Fig. 7a), indicating that Gal3 in HIPA contributes to the development of mechanical allodynia in gp120Tg mice. In addition, we performed Hargreaves tests to assess the role of Gal3 in thermal pain (Supplementary Fig. 10). Compared with WT mice, gp120Tg mice manifested significant increase of thermal pain sensitivity, and the thermal pain hypersensitivity was significantly attenuated by global Gal3 KO in the gp120Tg mice. In contrast to its effect on mechanical pain hypersensitivity, heterozygous KO of astrocytic Gal3 in gp120Tg mice did not significantly attenuate thermal pain, indicating astrocytic Gal3’s differential role in mediating the development of distinct pain modalities.

Since Gal3 is required for the formation of HIPAs (Fig. 6e–g, i), it is probable that Gal3 controls the expression of gp120-induced allodynia via HIPAs. How might HIPAs contribute to the pain pathogenesis initiated by gp120? Previous studies on HIV patient autopsies and animal models suggest that neurodegeneration, indicated by synaptic degeneration, is associated with HIV pain pathogenesis6,26,31. As HIPAs are neuronal phagocytes (Fig. 5), we determined the effect of Gal3 KO on neurodegeneration in the SDH of gp120Tg mice (5 months), by FJC staining to detect the degenerative neurons in WT, gp120Tg, and gp120Tg/Gal3−/− mice (5 months). Compared with WT spinal cords, gp120Tg spinal cords had significantly more FJC staining signals (Fig. 7b, c, e), indicating an increase of neurodegeneration. Gal3 KO blocked the increase of FJC signals in spinal cords of gp120Tg mice (Fig. 7d, e), suggesting Gal3 was involved in gp120-induced neurodegeneration in the SDH pain circuits.

Neuroinflammation in the SDH is a biomarker for pain development in HIV patients6, with activation of astrocytes being a hallmark specifically in HIV patients with chronic pain3. These findings suggest that HIPAs are probably among the reactive astrocytes that contribute to the pathogenic neuroinflammation underlying HIV-associated pain. Aligning with this hypothesis, snRNA-seq analysis revealed upregulated expression of inflammatory genes in HIPAs (Fig. 1k, Supplementary Fig. 2). To test this hypothesis, we compared protein levels of neuroinflammation markers in the spinal cords of WT, gp120Tg, and gp120Tg/Gal3−/− mice (5 months) using immunoblotting analysis (Fig. 7f). Specifically, we focused on neuroinflammation markers IL-1β, Wnt5a, and reactive microglia due to their confirmed contribution to the development of gp120-induced pain7.

Our results showed that cleaved mature IL-1β was upregulated in gp120Tg spinal cords, and this upregulation was abolished in gp120Tg/Gal3−/− mice (Fig. 7g). However, pro-IL-1β levels were not significantly different among WT, gp120Tg, and gp120Tg/Gal3−/− mice (Fig. 7h). These data suggest that Gal3 regulates IL-1β at the stage of its maturation rather than at transcription or translation stages. Since reactive astrocytes are the source of gp120-induced IL-1β7 and Gal3 KO diminished HIPA (Fig. 6g, i), we reasoned that Gal3 likely controls IL-1β maturation in HIPAs. To gain insight into the potential Gal3-regulated IL-1β activation mechanism, we tested the involvement of inflammasomes. We measured the levels of active-caspase1, which is the inflammasome-associated pro-IL-1β processing proteinase and observed that active-caspase1 was significantly upregulated in gp120Tg mice (Fig. 7f, i). Strikingly, Gal3 KO not only abolished the upregulation but also brought the active-caspase1 level drastically below the basal level (Fig. 7i). These results indicate that Gal3 regulates gp120-induced IL-1β via controlling the activation of inflammasomes in HIPAs.

Wnt5a is a regulator of neuroinflammation that is specifically elevated in the SDH of HIV-positive patients experiencing pain32 and plays a significant role in the pathogenesis of gp120-induced pain33. We observed that spinal Wnt5a was markedly upregulated in gp120Tg mice and that Gal3 KO significantly mitigated this increase in gp120Tg/Gal3−/− mice (Fig. 7j).

Reactive microglia are an important source of inflammatory mediators and critical for the expression of the early phase of gp120-induced pain7. In line with previous research26, we observed upregulation of the microglia/macrophage marker Iba1 in the spinal cord of gp120Tg mice (Fig. 7k), suggesting microglia activation. Importantly, we discovered that Gal3 KO attenuated the Iba1 upregulation (Fig. 7k), indicating an inhibitory effect of Gal3 KO on microglial activation.

Methods
Animal
All animal procedures were performed in compliance with protocol 0904031B approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, and in accordance with all relevant guidelines and regulations. Gp120Tg mice used in this study (provided by Dr. Marcus Kaul) express HIV-1 LAV gp120 under the control of the glial fibrillary acidic protein (Gfap) promoter15. Lgals3 Homozygotes KO (Gal3−/−) mice were obtained from the Jackson Laboratory (Strain: #006338). We received Lgals3-Flox mice57 from Dr. Danial Saban. Gfap-Cre mice were purchased from purchased from Jackson Laboratory (Strain: #024098). Mice were housed in a specific pathogen-free (SPF) facility at UTMB under controlled environmental conditions, including a 12-h light/dark cycle (light s on at 7:00 AM and off at 7:00 PM), ambient temperature maintained at 22 ± 2 °C, and relative humidity of 40–60%. Standard rodent chow and water were provided. Mice were anesthetized with 14% urethane or solfuorane. All primer sequences used for genotyping are included in the Supplementary Data 5. Male mice were used for snRNA-seq, western blot, injections, and immunofluorescent staining experiments. Mixed males and females were used for behavioral testing and RNAscope validation of the snRNA-seq data.

Lumbar spinal cord dissection
We performed spinal nuclei dissociation using the same tissue dissociation procedures for all experimental groups. To isolate nuclei for snRNA-seq, we euthanized WT, gp120Tg, gp120Tg/Gal3−/−, and Gal3−/− mice with 14% urethane followed by decapitation. We rapidly dissected out the lumbar spinal cords and temporarily stored them in 6 ml of cold Hibernate A/B27 (HABG) medium for further processing. HABG was prepared by adding 30 ml of Hibernate A (BrainBits, cat. no. HA), 600 µl of B27 (Thermo Fisher, cat. no. 17504044), 88 µl of 0.5 mM Glutamax (Invitrogen, cat. no. 35050-061), penicillin-streptomycin (Thermo Fisher, cat. no. 15070063) to a final concentration of 2%, and DNase I (Thermo Fisher, cat. no. AM2224) to a final concentration of 80 U/ml.

Nucleus dissociation from the lumbar spinal cord
We collected three lumbar spinal sections from each group and pooled them together for nuclei dissociation. To isolate nuclei for snRNA-seq, we used the detergent-mechanical cell lysis method58. In brief, the lumbar spinal sections were placed in a pre-chilled Dounce homogenizer, and 500 µl of pre-chilled detergent lysis buffer was added. The detergent lysis buffer was prepared by adding 3 µl of 20% Triton-X to 600 µl of low sucrose buffer (sucrose: 0.32 M; HEPES (pH=8.0): 10 mM; CaCl2: 5 mM; MgAc: 3 mM; EDTA: 0.1 mM; DTT: 1 mM). We homogenized the tissue first by 5 strokes with pestle A (‘loose’ pestle), then 5–10 strokes with pestle B (‘tight’ pestle) to prepare crude nuclei. We did not lift pestles out of the lysis buffer between strokes to minimize bubble creation during homogenization. We added 1 ml of low sucrose buffer to the Dounce homogenizer containing the crude nuclei and mixed gently by pipetting 2–3 times. We passed the crude nuclei preparation through a 40 µm strainer (Fisher scientific, cat. no. 08-771-1) into a pre-chilled 50 ml conical tube containing 1 ml of low sucrose buffer. Then, an additional 1 ml of low sucrose buffer was passed through the strainer, which brought the volume of the nucleus preparation to 3.5 ml (3 ml of low sucrose buffer and 500 µl of lysis buffer). We centrifuged the samples for 10 min (3200 × g; 4 °C), and resuspended the pellets using 3 ml of low sucrose buffer by gently swirling to wash the pellet off the wall. The suspension was put on ice for 2 min and then transferred to an Oak Ridge tube. We homogenized the suspended nuclei for 30 s on ice using a homogenizer (IKA, Ultra Turrax T8; at setting 1). We carefully added 12.5 ml of density sucrose buffer (sucrose: 1 M; HEPES (pH=8.0): 10 mM; MgAc: 3 mM; DTT: 1 mM) underneath the low sucrose buffer homogenate using a serological pipette to prevent creating any bubbles that could disrupt the density layers. We then centrifuged the Oak Ridge tube at 3200 × g for 20 min at 4 °C. Once the centrifugation was complete, we immediately decanted the supernatant in a flicking motion. The nuclei remaining on the wall were resuspended with 500 µl of resuspension solution (BSA: 0.4 mg/ml; RNAse Inhibitor: 0.2 U/µl). The sample was then passed through a 40 µm Flowmi pipette tip filter (Sigma-Aldrich, cat no: H13680-0040) before counting and loading to remove clumped nuclei and large debris that can clog the microfluidics of Chromium chips. We collected the filtered nuclei in a pre-chilled tube and determined the nucleus yield using a hemocytometer. The nuclei were immediately processed for the next step. Due to high myelin contents in spinal cords, the yield of nuclei was relatively low59.

3’ single-nucleus RNA library construction and sequencing
Gel Bead-In Emulsions (GEMs) were generated using the Chromium Platform from 10x Genomics (cat. no. PN-1000075) following the Chromium protocol CG000185 Rev. The Chromium Single Cell 3′ Library & Gel Bead Kit was used for GEM and library preparation. Briefly, 10,000 to 20,000 nuclei were loaded onto the Chromium controller to recover 7000–15,000 nuclei for library preparation and sequencing. GEMs were captured and underwent reverse transcription by incubating at 53 °C for 45 min in a thermal cycler. Next, GEMs were broken, and the sample cDNA was recovered and cleaned according to the instructions. A 35 µl sample of recovered cDNA was used for cDNA amplification using the reagents provided in the kit, and 12 PCR cycles were used. The amplified cDNA was cleaned and quantified using an Agilent Bioanalyzer High Sensitivity chip.

For generating the 3ʹ Gene Expression library, 10 µl of purified cDNA sample was enzymatically fragmented and size-selected using SPRIselect magnetic beads. Illumina sequencing adapters were then ligated to the size-selected fragments and cleaned up using SPRIselect magnetic beads. Sample indices were selected and amplified, followed by a double-sided size selection using SPRIselect magnetic beads. The final library quality was assessed using an Agilent Bioanalyzer High Sensitivity chip. The libraries were sequenced on the Illumina NovaSeq to target a minimum of ~15,000 reads per nucleus with a total of 300 million paired-end reads per sample. The library sequencing was performed at the Single Cell Genomics Core, Baylor College of Medicine, USA.

Seurat snRNA-seq integration analysis
The raw sequencing data were pre-processed using Cell Ranger (v.3.1.0) (10X Genomics). The resulting filtered single-cell expression matrices files from the WT, gp120Tg, gp120Tg/Gal3−/−, and Gal3−/− mice were imported into R for Seurat snRNA-seq integration analysis. Seurat R package (v.4.0.5) was utilized for dimension reduction and cluster analysis. Seurat implements a global-scaling normalization method “LogNormalize” by default that normalizes gene expression measurements for each nucleus by total expression, multiplies this by a scale factor (10,000 by default), and log-transforms the result. To remove low-quality nuclei and doublets, we set the filter criteria of nFeature_RNA > 200 and nFeature_RNA < 5000 as a QC control for snRNA-seq data. This was followed by normalization and selection of all highly variable genes falling within a selected cut-off window for PCA analysis. Statistically significant PCs (p-value < 0.001) were utilized in cluster determination. UMAP plots were produced at a resolution of 0.6, and clusters were annotated based on the expression level of canonical marker genes, and gene expression was visualized using feature maps. The astrocyte clusters were identified using the classic marker genes Gfap, Aqp4, and Gja1. The astrocyte clusters from the integrated UMAP plot were subset for astrocyte subset analysis. UMAP plots of astrocytes were produced at a resolution of 0.5. The integrated UMAP plot was split by genotype to view the astrocyte cluster at each condition.

Immunofluorescent (IF) staining
Mice were anesthetized with 14% urethane and transcardially perfused with 30 ml of cold 1× PBS. The L4–L5 lumbar spinal cord segments were collected, fixed in freshly prepared 4% paraformaldehyde for 24 h at 4 °C, and then immersed in 30% sucrose in 1× PBS at 4 °C until they sank to the bottom. The tissues were embedded in OCT mounting medium, stored in an air-tight container at –80 °C, and equilibrated to –20 °C in a cryostat for ~1 h prior to sectioning. Cryo-sections ranging from 10 to 35 µm were cut, mounted onto SuperFrost® Plus slides, or stored in Hito floating section storage solution (Hitobiotec) at –20 °C until they were stained for immunocytochemistry.

For immunostaining, sections were rinsed with 1× PBS twice to remove storage solution and blocked with 5% BSA and 0.3% Triton X-100 in 1× PBS for 2 h at room temperature. This was followed by 48 h of incubation with primary antibodies, including anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. no. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. no. 016–20001), ant-Vimentin antibody (1:500, Abcam, cat. no. ab137321), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), anti-Sox9 antibody (Sigma-Aldrich, cat. No. ab5535) and anti-vGlut1 antibody (Synaptic systems, cat. no. 35303). After five washes with PBS, the sections were incubated with the corresponding second antibodies at room temperature for 2 h. The stained sections were mounted with ProLong Gold Antifade Mountant (Invitrogen, cat. no. P36930) and stored overnight at 4 °C. Images were obtained using Zeiss LSM880 with Airyscan (Carl Zeiss Microscopy GmbH) and Nikon A1RMP (Nikon Instruments Inc.), and the 3-D images were processed using Imaris 9.6 software (Oxford Instruments).

Fluoro-Jade C (FJC) staining
10-µm cryo-sections of lumbar spinal cord were prepared for Fluoro-Jade C (FJC) staining to identify degenerating neurons. FJC staining was performed using the Fluoro-Jade C Ready-to-Dilute Staining Kit (Biosensis, cat. no. TR-100-FJ) following the manufacturer’s instructions. After FJC staining, immunofluorescence (IF) staining was performed on the same sections using the anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938) according to the protocol described above. The stained sections were mounted with ProLong Gold Antifade Mountant (Invitrogen, cat. no. P36930) and stored overnight at 4 °C. Images were visualized using Zeiss LSM880 with Airyscan (Carl Zeiss Microscopy GmbH) and Nikon A1RMP (Nikon Instruments Inc.).

RNAscope in situ hybridization (ISH) and quantification of C3+ astrocytes
Mice were anesthetized with isolfuorane then euthanized by decapitation. Spinal cords were collected and first fixed in fresh 4% PFA for 24 h at 4 °C. The spinal cords were then immersed in 15% sucrose solution at 4 °C for 12 h, then 30% sucrose solution at 4 °C for 12 h. Each spinal cord was then embedded in OCT mounting medium and stored at –80 °C prior to sectioning. The mounted tissue blocks were equilibrated to –20 °C in a cryostat, then 15 µm sections were cut and mounted onto SuperFrost® Plus slides and used immediately. Probes for the gene C3 (417841) were purchased from Advanced Cell Diagnostics (Newark, CA). RNAscope procedures, including tissue pre-treatment and probe hybridization, were performed according to the protocol of RNAscope® Multiplex Fluorescent V2 Assay provided by the manufacturer (Advanced Cell Diagnostics). After the last step of RNAscope, the sections were immunostained for Gfap and Lgals3 as described (Dual ISH-IHC, Advanced Cell Diagnostics). The stained sections were mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, cat. no. H-1500-10) and stored overnight at 4 °C before imaging using a Nikon AX confocal microscope. Anti-Gfap antibody (abcam, cat. ab134436) and Anti-Lgals3 Antibody (Santa Cruz Biotechnology, catalog no. sc-23938) were both used at 1:200 dilution. The images were acquired on a Nikon confocal microscope and analyzed using ImageJ (v1.54p). We quantified C3 signals within HIPAs (Gal3+) in gp120Tg mice and non-HIPA astrocytes (Gal3−) in wild-type, gp120Tg, and gp120Tg/gal3-KO mice and found that 3 pixels of C3 signals were an effective threshold to differentiate HIPAs from non-HIPA astrocytes. Using this cutoff value, we designed a computerized procedure to count C3+ astrocytes (GFAP+). To validate the data from this automatic procedure, we also asked two individuals without access to group information to count C3+/GFAP+ cells.

Postmortem human spinal tissues
Spinal cord specimens of HIV+ patients and non-infected controls were procured from the Texas NeuroAIDS Research Center (TNRC) of the National NeuroAIDS Tissue Consortium (NNTC). The research received oversight from the Institutional Review Board (IRB) of the University of Texas Medical Branch (UTMB) (Federal Wide Assurance FWA#00002729). The UTMB IRB approval number is 98-402. The authorization for autopsy was obtained from next-of-kin in accordance with Texas statutory law. Informed consent was obtained from decedents who were participants in a clinical research protocol when alive. Consent forms were reviewed by the IRB, date-stamped, and approved annually. Informed consent was obtained annually.

Western blotting analysis
To prepare mouse tissue, mice were euthanized using excess anesthesia (isoflurane), and L4–L5 lumbar spinal cord segments were collected. Spinal tissues were homogenized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, pH 8.0) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor mixture (Sigma-Aldrich, cat. no. P8340). The protein concentration in the homogenates was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, cat. no. 23227). The protein concentration was adjusted to 2 µg/µl for immunoblotting analysis. Equal amounts of protein (30 µg/lane) were loaded and separated by SDS-PAGE, followed by transferring to polyvinylidene difluoride membranes (Millipore, cat. no. IPVH00010).

For immunoblotting analysis, the blots were blocked by 5% nonfat milk in Tris-buffered saline–Tween-20 (20 mM Tris-HCl, 150 mM NaCl, pH 7.5, 0.1% Tween-20) for 2 h at room temperature, then sequentially incubated with primary and secondary antibodies. The primary antibodies used were anti-Gal3 (1:500, Santa Cruz, cat. no. sc-23938), anti-cleaved Il1β (1:500, Millipore, cat. no. ab1413-I), anti-pro-Il1β (1:500, Cell Signaling Technology, cat. no. 12242), anti-cleaved caspase-1 (1:1000, Cell Signaling Technology, cat. no. 4199), anti-wnt5a (1:1000, Abcam, cat. no. ab72583), anti-Iba1 (1:1000, Wako, cat. no. AB_839506), and anti-actin (1:1000, Santa Cruz, cat. no. sc-1616). The protein bands were visualized using the Pierce Enhanced Chemiluminescence Kit (Thermo Fisher Scientific, cat. no. 32106), with β-actin blotted as a loading control. The intensity of the bands was quantified using densitometry analysis with NIH ImageJ. Uncropped and unprocessed scans are provided in the source files.

Gp120 injection
Recombinant gp120 protein (Cat# HRP20082) was obtained through BEI Resources - a division of the National Institute of Allergy and Infectious Diseases. A 2 µL aliquot of the stock solution was diluted with 0.1% bovine serum albumin in 0.1 M phosphate-buffered saline for a final concentration of 15 ng/µL. Mice were initially anesthetized by inhaling 3% isoflurane. Once fully anesthetized, a 30-gauge needle was inserted at a 45° angle into the intervertebral space between L5 and L6. Correct needle placement was confirmed by a tail twitch from the anesthetized mouse. Finally, 7 µL of the gp120 solution was expelled at an approximate rate of 1 µL per 3 s. After delivery, the needle was held at the injection position for 15 s to ensure no spillage of solution. Subjects were injected every other day for a total of 3 injections per mouse.

Mechanical pain tests
To measure paw withdrawal thresholds (PWTs), von Frey testing was conducted on the plantar surface of the hind paw. Mice were habituated to the testing surroundings for 2 h per day for three days prior to the testing day. On the day of testing, calibrated von Frey filaments (ranging from 0.1 to 2.0 g) were applied perpendicularly to the central area of the hind paw. The Dixon “up and down paradigm” method was used to calculate the PWT values. To minimize subjective effects and bias, behavioral tests were performed in a double-blind manner, with the genotype of individual animals not revealed to the experimenters.

Thermal hypersensitivity tests
Thermal withdrawal latencies were measured using the Hargreaves test. For this, mice were placed in clear plastic chambers (7 × 9 × 10 cm) on an elevated surface and allowed to acclimatise to their environment before testing. The radiant heat source was aimed at the plantar surface of each hind paw in the area immediately proximal to the toes. The paw withdrawal latency (PWL) was defined as the time from the onset of radiant heat until a withdrawal of the hind paw occurred. A 20-s cutoff time was used to avoid tissue injury and sensitisation. Three measures of PWL were taken and averaged for each hind paw.

Statistical analysis
Based on our results from previous experiments, we calculated a cohen’s d of 2.08 between groups. To achieve an alpha of 0.05 and power of 80%, we use G*Power (v3.1) to determine that a minimum five animals were required. Further statistical analysis was conducted using Prism 5 software (GraphPad). Data are presented as mean ± SEM or mean ± SD. Immunoblotting data were analyzed using either Student’s t test or nonparametric Mann–Whitney test or One-way ANOVA. Immunofluorescent staining, FJ-C staining, and pain behavioral data were analyzed using a One-way ANOVA or Student’s t test. A p-value of less than 0.05 was considered statistically significant for all tests.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Zheng et al. investigated a novel cellular mechanism of HIV-induced neuropathic pain in HIV-1 gp120 transgenic mouse\nmodels. Using single nucleus RNA-seq (snRNA-seq) transcriptomic analysis, the authors identified a novel subtype of HIV\npain-associated astrocytes (HIPAs) in the lumbar spinal cord of HIV-1 gp120 transgenic mice and also in the spinal cord of\nHIV patients. They showed that HIPAs express galectin 3 (Gal3) and have transcriptomic signatures of phagocytosis and\ninflammation. They also showed some evidence of phagocytosis of spinal cord neurons by HIPAs. Importantly, they showed\nthat knockout (KO) of Gal3 in gp120 transgenic mice reduced the number of HIPAs, mechanical allodynia, and neuronal\ndegeneration and neuroinflammation. The findings are very novel, especially the identification of cluster 7 in the gp120Tg\nmice. There is also strong evidence for a critical contribution of Gal3 to HIV-induced pain and neuroinflammation. Adding\nspinal cord samples of HIV patients increased translational relevance. The manuscript is well written. However, the\nfollowing issues should be addressed to support the major conclusion of this study.\nMajor:\n1. It is unclear if Gal3 in astrocytes is sufficient to regulate HIV-associated pain. This study identified Gal3 (Lgals3) is highly\nexpressed in astrocytic subtype cluster 7). Galectin 3 is also known to be expressed in spinal microglia in pain regulation.\nGal3 global KO mice do not tell a specific role in astrocytes. It will strengthen the paper if the authors can specifically\nknockdown Lgals3 in astrocytes of gp120-treted mice using astrocyte-specific virus vectors.\n2. Line 95: 20,284 nuclei were pooled from three wild-type (WT) and three gp120Tg mice. This yield is not so high. It is\nstated that HIPAs comprised 20% of total gp120Tg spinal astrocytes. The authors only showed the relative distribution of 8\nastrocyte clusters between WT and gp120Tg groups. It will be helpful to show the cell number in each cluster so that readers\ncan see changes of astrocyte numbers in different clusters after HIV pathology.\n3. Fig. 2c: Approximately 95% of the Gal3+ cells were also GFAP+. However, the number of Gal3+ cells appears to be low in\nthe spinal cord.\n4. The authors named this astrocytic subtype (Cluster 7) as HIV-pain associated astrocytes (HIPAs). I wonder if you also see\nthis cluster in other neuropathic pain condition(s).\nMinor:\nA1 and A2 astrocytes were mentioned in introduction but not in results and discussion. Which cluster of astrocytes in\ngp120Tg spinal cord may represent A2 astrocytes?\nFigure 4. HIPAs engulfed neuronal soma in the gp120Tg spinal cord. Are these excitatory or inhibitory neurons? This may\nresult in different changes in pain.", "reviewer_2": "In this study, the authors define an astrocyte subtype, they termed HIV-pain associated astrocytes (HIPAs), which expresses\nGal3 and has a distinct transcriptomic signatures of phagocytosis and inflammation. KO of Gal3 depleted this cell type and\nattenuated neuronal degeneration and neuroinflammation. The authors conclude that HIPAS contribute to gp120-induced\npain through inducing neuroinflammation and neurodegeneration in spinal pain neuronal circuit.\nThe authors present (for the most part) well controlled studies that shows the role of Gal3+ astrocytes in gp120-mediated\nneuropathy, modeled in mice. The major concern with this study is regarding perhaps overreach and over interpretation that\nthese cells have a distinct novel lineage that is induced in response to gp120 in this model. Much of what is modeled in Fig.\n7 is speculative with no specific studies to address that HIPA is a novel new lineage specific population rather than GAL3+\nexpression is induced on reactive astrocytes and GAL3 then promotes neuroinflammation as was described previously. As\nsuch the scientific premise that Gal3-expressing astrocytes is a unique subtype of astrocytes related to HIV-associated pain\nis rather questionable because those cells may not be a separate lineage (as the title suggests) but rather responsive cells\nto this type of stimuli, which makes the title misleading. More data is needed to prove the claim that this is a lineage-specific\npopulation\nIt is unclear if these cells are responsive to pain-related signals in general or just to HIV-specific signals? To make this\ngeneralization, a non-HIV pain-related animal model would be helpful to determine if this population is relevant to HIV only\nor other pain models/systems. Otherwise, that claim should be reassessed\nMuch of the experiments rely on WT vs. gp120 Tg mice comparisons, a scrambled gp120 or irrelevant similar size protein\nwould be helpful to assess presence (generation) of HIPAs and responsive to neuropathic pain", "reviewer_3": "In this interesting article by Zheng and colleagues HIV-Pain Associated Astrocytes (HIPA) are examined and the authors\nidentify a specific HIPA population that is marked by Galectin 3. The paper starts out using scRNA-seq from the spinal cords\nof HIPA mouse models and identifies striking differences in the astrocyte constituency. Further analysis identifies Galectin 3\nas a prospective marker of this HIPA population. Functional studies ensue, where they characterize the origin of this\npopulation, its possible functional interactions with neurons, and the role Galectin 3 in pain responses.\nOverall, the broad idea of this manuscript is quite enticing as the authors claim to have identified a specific subpopulation of\nastrocytes linked to HIPA and then perform a series of functional studies with that population. However, the experiments are\nvery poorly conducted, the data is of very poor quality, and the conclusions are specious (at best). Accordingly, this\nmanuscript does not meet the standard for publication in Nature Communications. Below are a series of specific comments\nand suggestions that I hope the authors will take under advisement.\n1) The images are very poor quality—particularly figures 3, 4, and 6. Some of these images are out of focus and figures 3-4\nhave no quantification. Also Figure 2 needs more quantification---specifically what percentage of astrocytes express\nGalectin 3? This could be performed with a Sox9 co-stain.\n2) The notion that these Galectin3+ HIPA’s are involved in neuronal degeneration or englufment is enticing and interesting.\nHowever the data in figure 4 does not prove this, nor even come close to proving this. To prove this would require in vitro\nsystems, along with some high resolution, in vivo imaging using new viral tools generated by Won-Suk Chung’s lab to\nmeasure this in vivo (Lee, et al. Nature 2021). These are tedious and time consuming assays, that are often also\ninconclusive. Moreover, the evidence that astrocytes engluf neurons during such disease states is unreliable, at best.\n3) The claim that the Galectin 3 astrocytes are emanating from the central canal is also interesting, but it not at all proven.\nThe images in figure 3 are like the opening argument—but to prove this hypothesis, they need to lineage trace cells\nspecifically from the central canal. This would require a Galectin 3-CreER line or some other line that maker from the central\ncanal. Accordingly, another model that explains this phenomenon is that Galectin 3 is simply turned on by astrocytes in the\nparenchyma.\n4) The Galectin-KO studies are interesting----however they are lacking the Galectin-KO control (without the gp120Tg).\nMoreover, the phenotype in VonFrey is very weak and is almost non-existent. Furthermore, this experiment is really\nunderpowered (some only have three mice). Finally, the authors should conduct a comprehensive battery of pain-related\nbehaviors. Given the mild effect with Von Frey, I would guess that the Galectin-KO;gp120Tg probably didn’t have any other\npain-related phenotypes.\n5) The whole A1/A2 nomenclature is no longer valid and should be removed from the introduction and the paper entirely.\nSee this review (Escartin, et at. Nature Neuroscience 2021), as nearly the entire astrocyte field has come to an agreement\nabout this."}, "author_response": "Response to the Reviewer’s Comments\nReviewer #1 (Remarks to the Author):\nZheng et al. investigated a novel cellular mechanism of HIV-induced neuropathic\npain in HIV-1 gp120 transgenic mouse models. Using single nucleus RNA-seq\n(snRNA-seq) transcriptomic analysis, the authors identified a novel subtype of HIV\npain-associated astrocytes (HIPAs) in the lumbar spinal cord of HIV-1 gp120\ntransgenic mice and also in the spinal cord of HIV patients. They showed that\nHIPAs express galectin 3 (Gal3) and have transcriptomic signatures of\nphagocytosis and inflammation. They also showed some evidence of phagocytosis\nof spinal cord neurons by HIPAs. Importantly, they showed that knockout (KO) of\nGal3 in gp120 transgenic mice reduced the number of HIPAs, mechanical allodynia,\nand neuronal degeneration and neuroinflammation. The findings are very novel,\nespecially the identification of cluster 7 in the gp120Tg mice. There is also strong\nevidence for a critical contribution of Gal3 to HIV-induced pain and\nneuroinflammation. Adding spinal cord samples of HIV patients increased\ntranslational relevance. The manuscript is well written. However, the following\nissues should be addressed to support the major conclusion of this study.\nResponse: We are grateful for the reviewer’s overall enthusiasm on our findings.\nMajor:\n1. It is unclear if Gal3 in astrocytes is sufficient to regulate HIV-associated pain.\nThis study identified Gal3 (Lgals3) is highly expressed in astrocytic subtype cluster\n7). Galectin 3 is also known to be expressed in spinal microglia in pain regulation.\nGal3 global KO mice do not tell a specific role in astrocytes. It will strengthen the\npaper if the authors can specifically knockdown Lgals3 in astrocytes of gp120-\ntreted mice using astrocyte-specific virus vectors.\nResponse: We appreciate the reviewer's insightful suggestion. Because Gal3+ reactive\nastrocytes (HIPAs) constitute only a small portion of total astrocytes [6% based on\nsnRNA-seq data (Supplementary Table 4) or ~2% (Supplementary Figure 4) based on\nGal3+Sox9+ co-staining – see response #2 to Reviewer #3)], we felt that targeting these\nsparsely distributed cells to knockdown Gal3 could be challenging for a viral approach.\nHence, we decided to take a more robust but much more time-consuming approach of\nconditional knockout (CKO) of Gal3 in astrocytes. We went through the lengthy processes\nof obtaining floxed Gal3 mice from another institution and setting up breeding trying to\nraise the gp120Tg/GFAP-Cre/Gal3flox/flox mice. This endeavor turned out to be super\nambitious because bringing the genes into the same mouse is not only very time\nconsuming but also with very low productivity. After more than a year of busy breeding,\nwe have only obtained a small number of heterozygous conditional knockout (CKO) mice\n(gp120Tg/GFAP-Cre/Gal3flox/+) that matured to 4-5 months for behavioral testing.\nSignificantly, to our delight, we found that, like global KO of Gal3, heterozygous CKO of\nGal3 in astrocytes significantly attenuated the expression of mechanical hypersensitivity\n1\nof gp120Tg mice (Fig. 7a in the revised manuscript). This new data is described in\ndescribed in RESULTS (page 7, paragraph 3).\n2. Line 95: 20,284 nuclei were pooled from three wild-type (WT) and three gp120Tg\nmice. This yield is not so high. It is stated that HIPAs comprised 20% of total\ngp120Tg spinal astrocytes. The authors only showed the relative distribution of 8\nastrocyte clusters between WT and gp120Tg groups. It will be helpful to show the\ncell number in each cluster so that readers can see changes of astrocyte numbers\nin different clusters after HIV pathology.\nResponse: The number of 20,284 nuclei is not the total yield of dissociated nuclei from\nthe pooled samples. Instead, this number represents the sum of the nuclei from the two\nsamples (including WT and gp120Tg) loaded onto 10x Chromium Controller that\nunderwent sequencing, passed the quality control filter and subjected to snRNA-seq\ncluster analysis. We apologize for not describing this clearly in the previous manuscript.\nWe have re-addressed this number in the revised manuscript (page 2, paragraph 4).\nWe have now included the number of astrocytes in individual clusters in Supplementary\nTable 1 and added this information to the main text (page 3, paragraph 2) in the revised\nversion.\n3. Fig. 2c: Approximately 95% of the Gal3+ cells were also GFAP+. However, the\nnumber of Gal3+ cells appear to be low in the spinal cord.\nResponse: We apologize for the confusion in the description. Fig. 2c is the quantitative\nsummary based on IF data representatively shown in Fig. 2a and 2b, to show that of all\nthe Gal3+ cells, 95% of them are astrocytes, while the remaining 5% Gal3+ cells are non-\nastrocytic cells (microglia). However, within the spinal astrocyte population of gp120Tg\nmice, Gal3+ astrocytes constitute only a small portion, as suggested by snRNA-seq data\n(Supplementary Table 4 in the revised manuscript). That was why Gal3+ cells revealed\nby IF appeared sparse the spinal cord. We have revised RESULTS (page 4, paragraph\n4) and Fig. 2c legends to clarify this.\n4. The authors named this astrocytic subtype (Cluster 7) as HIV-pain associated\nastrocytes (HIPAs). I wonder if you also see this cluster in other neuropathic pain\ncondition(s).\nResponse: We thank the reviewer for the important comment. Interestingly, a similar\nGal3+ astrocytic subtype (named Mac2 reactive astrocytes) was reported in the spinal\ncord of amyotrophic lateral sclerosis (ALS) mouse models. Like HIPAs, Mac2 cells also\nexpressed other astrocytic markers, such as GFAP, ALDH1L1, and S100β, but not\nmicroglial markers1,2. Mac2 cells also manifested morphologies similar to HIPAs,\nincluding large round cell bodies and relatively short processes. As ALS patients also\ndevelop pain, it will be significant to test if Mac2 cells also contribute to ALS-related pain.\nWe have revised DISCUSSION to compare HIPAs and Mac2 cells and consider the\npossible contribution of HIPA-like cells to ALS pain pathogenesis (Page 10, paragraph 4).\nMinor:\n5. A1 and A2 astrocytes were mentioned in the introduction but not in the results and\n2\ndiscussion. Which cluster of astrocytes in gp120Tg spinal cord may represent A2\nastrocytes?\nResponse: Notwithstanding controversies regarding A1 and A2 classification (see #6\nResponse to Reviewer #3), we mapped the detected A1 and A2 marker genes3,4 to\nastrocyte clusters. Our analysis revealed that both complement 3 (C3), a marker for A1\nastrocytes, and Cd109, a marker for A2 astrocytes, are highly expressed in clusters 7 and\n3 (Fig.1k, Fig. 6s and supplementary data Figure 10). However, we did not detect the\nexpression of other commonly used marker genes (e.g., Gbp2, Ptx3) for A1 and A2\nastrocytes. We have revised DISCUSSION (page 11, paragraph 3) to consider these new\nfindings.\n6. Figure 4. HIPAs engulfed neuronal soma in the gp120Tg spinal cord. Are these\nexcitatory or inhibitory neurons? This may result in different changes in pain.\nResponse: Thank you for this insightful question. In a separate study to be published\nelsewhere, we conducted immunofluorescent staining and 3D image analysis of GFAP,\nGal3, and excitatory or inhibitory synaptic markers. Our data showed that Gal3+\nastrocytes contained excitatory synapses but not inhibitory synapses in both cortices and\nspinal cords of gp120Tg mice, suggesting HIPAs selectively engulf excitatory neurons.\nNonetheless, we have revised DISCUSSION to acknowledge that HIPAs’ potential\nselectivity in phagocytosis is an unsolved issue in the current study (page 10, paragraph\n2).\nReviewer #2 (Remarks to the Author):\nIn this study, the authors define an astrocyte subtype, they termed HIV-pain\nassociated astrocytes (HIPAs), which expresses Gal3 and has a distinct\ntranscriptomic signature of phagocytosis and inflammation. KO of Gal3 depleted\nthis cell type and attenuated neuronal degeneration and neuroinflammation. The\nauthors conclude that HIPAS contributes to gp120-induced pain through inducing\nneuroinflammation and neurodegeneration in spinal pain neuronal circuit.\n1. The authors present (for the most part) well controlled studies that show the role\nof Gal3+ astrocytes in gp120-mediated neuropathy, modeled in mice. The major\nconcern with this study is regarding perhaps overreach and over interpretation that\nthese cells have a distinct novel lineage that is induced in response to gp120 in\nthis model. Much of what is modeled in Fig. 7 is speculative with no specific studies\nto address that HIPA is a novel new lineage specific population rather than GAL3+\nexpression is induced on reactive astrocytes and GAL3 then promotes\nneuroinflammation as was described previously. As such the scientific premise\nthat Gal3-expressing astrocytes is a unique subtype of astrocytes related to HIV-\nassociated pain is rather questionable because those cells may not be a separate\nlineage (as the title suggests) but rather responsive cells to this type of stimuli,\nwhich makes the title misleading. More data is needed to prove the claim that this\nis a lineage-specific population.\nResponse: We appreciate the thoughtful and constructive comments. The objective of\nthis work was to identify cell subtypes related to pain pathogenesis induced by HIV-1\ngp120, based on clustering analysis of snRNA-seq data, and we think that our results\nsuggest that HIPA is such a subtype of reactive astrocytes. However, we agreed with the\nreviewer on the potential risk of being overinterpreting the available data on that HIPA\nwas derived from a novel cell lineage. We have made the following revisions to improve\naccuracy of interpretation: 1). We deleted the original Fig. 7 (the model) in the previous\nmanuscript, 2) We revised the title and the whole MS to emphasize HIPAs as a subtype\nof reactive astrocytes, 3) when discussing the possibility of HIPA being derived from\nependymal cells in DISCUSSION, we clearly stated alternative possibilities and the need\nof direct evidence for validation of our idea (page 11, paragraph 2). An author of this paper\n(AD, a PhD student in the Tang lab) is continuing on this line of investigation to directly\ntest the ependymal hypothesis of HIPA origin; her studies are out of the scope of this\npaper and will be reported separately in the future.\n2. It is unclear if these cells are responsive to pain-related signals in general or just\nto HIV-specific signals? To make this generalization, a non-HIV pain-related animal\nmodel would be helpful to determine if this population is relevant to HIV only or\nother pain models/systems. Otherwise, that claim should be reassessed.\nResponse: We are grateful to the reviewer for this insightful comment. We think that this\ncomment is similar to Reviewer 1’s #4 comment and has been responded there.\n3. Much of the experiments rely on WT vs. gp120 Tg mice comparisons, a\nscrambled gp120 or irrelevant similar size protein would be helpful to assess\npresence (generation) of HIPAs and responsive to neuropathic pain.\nResponse: Thanks for this thoughtful comment. In response, we performed a control\nexperiment to compare the effect of native gp120 with heat-denatured gp120. We chose\nto use heat-denatured gp120, instead of scrambled gp120 or irrelevant similar size protein,\nbecause we thought that the denatured form of gp120 would be the most relevant and\nspecific control for gp120 and we routinely used this negatively control for gp120-induced\npain. The result showed that compared to the vehicle-injected group, gp120 but not the\nboiled gp120 induced a significant increase of Gal3 expression in the lumbar spinal cord\n(Supplementary Figure 5). Because inactive gp120 failed to induce pain5, these findings\nsuggest gp120-induced HIPAs are associated with the pathogenesis of gp120-induced\npain. We have described this new data to RESULTS (page 5, paragraph 1).\nReviewer #3 (Remarks to the Author):\nIn this interesting article by Zheng and colleagues HIV-Pain Associated Astrocytes\n(HIPA) are examined and the authors identify a specific HIPA population that is\n4\nmarked by Galectin 3. The paper starts out using scRNA-seq from the spinal cords\nof HIPA mouse models and identifies striking differences in the astrocyte\nconstituency. Further analysis identifies Galectin 3 as a prospective marker of this\nHIPA population. Functional studies ensue, where they characterize the origin of\nthis population, its possible functional interactions with neurons, and the role\nGalectin 3 in pain responses.\nOverall, the broad idea of this manuscript is quite enticing as the authors claim to\nhave identified a specific subpopulation of astrocytes linked to HIPA and then\nperform a series of functional studies with that population. However, the\nexperiments are very poorly conducted, the data is of very poor quality, and the\nconclusions are specious (at best). Accordingly, this manuscript does not meet the\nstandard for publication in Nature Communications. Below are a series of specific\ncomments and suggestions that I hope the authors will take under advisement.\n1. The images are very poor quality—particularly figures 3, 4, and 6. Some of these\nimages are out of focus and figures 3-4 have no quantification.\nResponse: To address the comments, we have revised the relevant figures to\nimprove their quality as following:\nFigure 3:\nFigure 3 was a combination of low and high magnification images. The low magnification\nimages (Figures 3b, 3c and 3d in the previous manuscript) captured using 10x and 20x\nobjectives allow us to view the overall distribution of Gal3+ cells in the entire spinal cord.\nWe now included higher power images (Figures 3b, 3c, 3e in the revised manuscript) to\nprovide better views of the distribution of Gal3+ cells. Additionally, in the revised\nmanuscript, we added a higher magnification insert of the selected area near the central\ncanal of the gp120Tg mice in Figure 3e (previously Figure 3d), to better show the\ncolocalization of Gal3 with astrocytic marker proteins GFAP and Vimentin and the\ntrajectory of HIPAs.\nFigure 5:\nWe acquired super-resolution images of co-staining of markers of HIPAs (Gal3), neurons\n(NeuN), and astrocyte (GFAP) to better show HIPA-neuron interaction, using ZEISS\nAiryscan confocal super-resolution microscopy. Moreover, we made two 3D videos to\nillustrate neuronal components engulfed by a HIPAs (Supplementary video 1 and 2).\nFigure 5l (original Fig.6b).\nCombination of Fluoro-Jade C (FJC) staining and immunofluorescent staining of Gal3\nwas done to show the interaction between HIPAs and degenerating neurons. FJC staining\nreq\nh\nu ires potais sium perjm anganate pretreatment to reduce background. However,\n5\npotassium permanganate is a strong oxidant that can partially destroy tissue antigens\nand affect immunofluorescent staining. We optimized the potassium permanganate\npretreatment conditions for FJC staining followed by subsequent Gal3\nimmunofluorescence staining. Despite our efforts, the potassium permanganate still\nseemed to cause some problems for Gal3 staining, which may have contributed to the\nrelatively poor images. Nevertheless, the images convincingly showed colocalization of\nFJC and Gal3 signals. In the revised manuscript, we moved the previous Figure 6b to\nFigure 5m.\nQuantification:\nFigure 3 (original Fig. 3).\nIn the revised Fig. 3, we have included quantification of Gal3 signals (Fig. 3d).\nFigure 5 (original Fig.4).\nIn the revised Fig. 5, we have included quantification of Gal3+ astrocyte with phagocyted\nneuronal components (Fig. 5l). We pointed out this caveat in RESULTS (page 6;\nparagraph 4) and considered Dr. Chung’s viral tools as a potential alternative approach\nfor more efficient quantification in DISCUSSION (Page10; paragraph 2).\n2. Figure 2 needs more quantification---specifically what percentage of astrocytes\nexpress Galectin 3? This could be performed with a Sox9 co-stain.\nWe thank the reviewer for this suggestion. Accordingly, we performed co-staining of\nGFAP and Gal3 with Sox-9 (Supplementary Figure 4). The results showed that\nGal3+Sox9+ astrocytes were barely detectable in the WT spinal cord. They increased to\n~2% of Sox9+ astrocytes in the gp120Tg mice (p-value=0.0045). The new data of Sox9\nco-staining confirmed Gal3+ astrocytes induced in the gp120Tg mice. We have revised\nRESULTS to include the new data (page 5, paragraph 1).\n3. The notion that these Galectin3+ HIPA’s are involved in neuronal degeneration\nor englufment is enticing and interesting. However, the data in figure 4 does not\nprove this, nor even come close to proving this. To prove this would require in\nvitro systems, along with some high resolution, in vivo imaging using new viral\ntools generated by Won-Suk Chung’s lab to measure this in vivo (Lee, et al.\nNature 2021). These are tedious and time-consuming assays, that are often also\ninconclusive. Moreover, the evidence that astrocytes engulf neurons during such\ndisease states is unreliable, at best.\nResponse. We thank the reviewer's expert suggestion. The viral tools developed by\nChung’s lab to measure in vivo phagocytosis of synapses are indeed elegant. However,\nas noted by the reviewer, these assays are very time-consuming and often inclusive;\nwe considered they are out of the scope of the work at the current stage. Thus, we\ndirected our effort to demonstrate more convincingly HIPA’s engulfment of neuronal soma by\nsupper-resolution imaging. In the revised manuscript, we included 3D Z-stack images\ntaken by ZEISS Airyscan confocal super-resolution microscopy. Using Imaris AI software,\nwe created opaque solid surface of neurons and HIPAs. After removing the signals not\ninteracting with HIPAs, we clearly saw the neuronal somas enwrapped/engulfed by HIPAs\n(Fig. 5c, 5d, 5f). We also showed neuronal debris inside the HIPAs (Fig. 5k). In addition,\nwe added two 3D videos to illustrate neuronal somas enwrapped by HIPAs and the\nneuronal debris inside a HIPA (Supplementary video 1 and video 2). We feel that these\nimaging data onvincingly show HIPA’s engulfment of neurons in the spinal cord of gp120\nmice. Notherless, we appreciate, with careful consideration, the expert’s insighht from the\nreviewer and revised DISCUSSION to acknowledge that the engulfment findings need to\nbe confirmed in the future with a second approach, such as the viral labeling approach\ndeveloped by Dr. Chung (p10, paragraph2).\n4. The claim that the Galectin 3 astrocytes are emanating from the central canal is\nalso interesting, but it not at all proven. The images in figure 3 are like the opening\nargument—but to prove this hypothesis, they need to lineage trace cells\nspecifically from the central canal. This would require a Galectin 3-CreER line or\nsome other line that maker from the central canal. Accordingly, another model that\nexplains this phenomenon is that Galectin 3 is simply turned on by astrocytes in\nthe parenchyma.\nResponse. We are grateful for the thoughtful and constructive comment, which is well\ntaken. We consider this comment is like Reviewer #2’s comment #1 and have responded\nthere.\n5. The Galectin-KO studies are interesting----however they are lacking the Galectin-\nKO control (without the gp120Tg). Moreover, the phenotype in Von Frey is very\nweak and is almost non-existent. Furthermore, this experiment is really\nunderpowered (some only have three mice). Finally, the authors should conduct a\ncomprehensive battery of pain-related behaviors. Given the mild effect with Von\nFrey, I would guess that the Galectin-KO; gp120Tg probably didn’t have any other\npain-related phenotypes.\nResponse: The point is well taken. In response to the comment, we did the following: 1)\nincreased the number of mice in each group and reconducted the pain behavioral tests,\n2) added the Gal3 KO control, and 3) added new Hargeave testing. In addition, we also\nadded a new group of mice of heterozygous KO of astrocytic Gal3 in the gp120Tg mice.\nThe new data were presented in revised Fig, 7a and Supp Fig. 9.\nFor mechanical pain, our previous conclusions stayed true and confirmed by the new data,\nnamely, there is a significant increase in mechanical pain sensitivity in the gp120Tg mice\nand the pain hypersensitivity was significantly attenuated in by globally KO of Gal3\n(gp120Tg/Gal3-/-). In addition, mice with heterozygous KO of astrocytic Gal3\n7\n(gp120Tg/GFAP-Cre/Gal3+/-) also showed attenuated pain hypersensitivity. The section\nof RESULTS was revised to describe the data (page 7, paragraph 3). We agree with the\nreviewer that the mechanical pain hypersensitivity manifested by gp120Tg mice was not\nvery drastic. Compared with wild-type mice, gp120Tg mice had a 38.6% drop in mean\nwithdrawal threshold. Nevertheless, the difference reached statistical significance and\nwas repeatable by different team members under blinded conditions.\nFor thermal pain measured by Hargreave testing (Supplementary Figure 9), gp120Tg\nmice also manifested hypersensitivity and this thermal pain hypersensitivity was\nsignificantly attenuated by global Gal3 KO. However, heterozygous KO of astrocytic Gal3\nin the gp120Tg mice did not significantly attenuate thermal pain. We revised RESULTS\nto report the new data (page 7, paragraph 3).\n6. The whole A1/A2 nomenclature is no longer valid and should be removed from\nthe introduction and the paper entirely. See this review (Escartin, et at. Nature\nNeuroscience 2021), as nearly the entire astrocyte field has come to an agreement\nabout this.\nResponse: We have revised the MS accordingly (please see also #5 Response to\nReviewer #2). In fact, we found that HIPAs expressed both ‘A1’ and ‘A2’ markers,\nindicating that HIPAs cannot simply be classified as either A1 or A2 subtypes according\nto the binary scheme of astrocytic subtypes. We considered this finding in revised\nDISCUSSION (page 11, paragraph 3).\nReferences\n1. Trias, E., Barbeito, L. & Yamanaka, K. Phenotypic heterogeneity of astrocytes in motor neuron\ndisease. Clin Exp Neuroimmunol 9, 225-234 (2018).\n2. Komine, O., et al. Innate immune adaptor TRIF deficiency accelerates disease progression of ALS\nmice with accumulation of aberrantly activated astrocytes. Cell Death Differ 25, 2130-2146 (2018).\n3. Liddelow, S.A., et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature\n541, 481-487 (2017).\n4. Addis, D.R., DeBerry, J.J. & Aggarwal, S. Chronic Pain in HIV. Mol Pain 16, 1744806920927276\n(2020).\n5. Yuan, S.B., et al. Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.\nAnn Neurol 75, 837-850 (2014).\nReviewer #1 (Remarks to the Author):\nThe authors have significantly improved the manuscript by testing the CKO mice. They\nfound that heterozygous CKO of Gal3 significantly attenuated the expression of\nmechanical hypersensitivity of gp120Tg mice (Fig. 7a). They also addressed the issue\nof A1/A2 astrocytes raised by the third reviewer. Overall, this study is a significant\ncontribution to the field of glial biology and pain.\nResponse: We appreciate the reviewer’s support.\nReviewer #3 (Remarks to the Author):\nThe authors have taken time and great effort to revise their manuscript, however there\nare still several flaws with this study.\n1) While I appreciate the high resolution imaging that suggests that these HIV\nassociated astrocytes are phagocytic, in the absence of functional studies\ndemonstrating this, I remain unconvinced. The comparison between Wt and gp120\nmutant is not convincing, either some sort of in vitro study or viral tool or astrocyte-\nspecific Gal3-KO or KO of phagocytic receptors in Gal3+ astrocytes is very much\nneeded here. Otherwise, it's just a correlation and there is no definitive proof of\nphagocytosis.\nResponse: We appreciate the reviewer’s thoughtful comment, which is well taken.\nHowever, we feel the suggested studies on phagocytic mechanism mediated by specific\nphagocytic receptors are beyond the scope of the current work, as stated in the title. To\naddress the reviewer’s comment, we have revised DISCUSSION to consider this\nimportant line of potential future investigation. (2nd paragraph/p10)\n2) There is absolutely no reason to mention or include any mention of the A1/A2\nnomenclature. Any mention of this biology should really be eliminated, unless there is a\nvalid reason for including it---which perhaps I have missed\nResponse: revised accordingly.\n3) I also don't think that the authors can definitely say that they have identified a bona\nfide \"subtype\" of astrocyte in this study. To do so would require a direct comparison of\nfunctional properties with other astrocyte \"subtypes\" or populations in their model and\nshow that this population has unique functional properties. Accordingly, I think the\nphrasing of this manuscript needs to be modified and reframed to simply say that they\nare describing a population of astrocytes in HIV associated pain models. The notion that\nthe have a \"subtype\" is handwaving and really more appropriate for the discussion.\nResponse: Revised accordingly, by changing ‘subtype’ to ‘population’.\n4) The single cell data in Fig 6 is interesting, but needs to be independently validated.\nThey claim at the end of the associated results section that KO of Gal3 eliminates these\nHIV associated astrocytes. If this indeed the case, then they need to indendently\nvalidate that this population is absent in their Gal3-KO\nResponse: We appreciate this very specific and relevant comment. To validate HIPA,\nthe best marker we knew was Gal3, but it would not be available after its KO. So we\nwent through a time-consuming approach to screen a new marker suitable for\nimmunostaining (IF) or RNAscope. Based on our snRNA-seq data, we tested several\ncandidates and found one of them, C3 RNA, which was relatively enriched in HIPA,\ncould be detected by RNAscope. Using C3 as a HIPA marker, we found that spinal HIPA\nnumbers increased in the gp120 model but returned to baseline following Gal3 KO,\nsuggesting Gal3 KO eliminate HIPA induced by gp120. These new data are presented\nin Supplemental Fig. 9 and described in RESULTS (2nd paragraph/p7)."}, {"round": 2, "reviewer_comments": {"reviewer_3": "RESPONSE TO THE REVIEWER’S COMMENTS\nReviewer #1 (Remarks to the Author):\nZheng et al. investigated a novel cellular mechanism of HIV-induced neuropathic\npain in HIV-1 gp120 transgenic mouse models. Using single nucleus RNA-seq\n(snRNA-seq) transcriptomic analysis, the authors identified a novel subtype of HIV\npain-associated astrocytes (HIPAs) in the lumbar spinal cord of HIV-1 gp120\ntransgenic mice and also in the spinal cord of HIV patients. They showed that\nHIPAs express galectin 3 (Gal3) and have transcriptomic signatures of\nphagocytosis and inflammation. They also showed some evidence of phagocytosis\nof spinal cord neurons by HIPAs. Importantly, they showed that knockout (KO) of\nGal3 in gp120 transgenic mice reduced the number of HIPAs, mechanical allodynia,\nand neuronal degeneration and neuroinflammation. The findings are very novel,\nespecially the identification of cluster 7 in the gp120Tg mice. There is also strong\nevidence for a critical contribution of Gal3 to HIV-induced pain and\nneuroinflammation. Adding spinal cord samples of HIV patients increased\ntranslational relevance. The manuscript is well written. However, the following\nissues should be addressed to support the major conclusion of this study.\nReviewer #2 (Remarks to the Author):\nIn this study, the authors define an astrocyte subtype, they termed HIV-pain\nassociated astrocytes (HIPAs), which expresses Gal3 and has a distinct\ntranscriptomic signature of phagocytosis and inflammation. KO of Gal3 depleted\nthis cell type and attenuated neuronal degeneration and neuroinflammation. The\nauthors conclude that HIPAS contributes to gp120-induced pain through inducing\nneuroinflammation and neurodegeneration in spinal pain neuronal circuit.\n1. The authors present (for the most part) well controlled studies that show the role\nof Gal3+ astrocytes in gp120-mediated neuropathy, modeled in mice. The major\nconcern with this study is regarding perhaps overreach and over interpretation that\nthese cells have a distinct novel lineage that is induced in response to gp120 in\nthis model. Much of what is modeled in Fig. 7 is speculative with no specific studies\nto address that HIPA is a novel new lineage specific population rather than GAL3+\nexpression is induced on reactive astrocytes and GAL3 then promotes\nneuroinflammation as was described previously. As such the scientific premise\nthat Gal3-expressing astrocytes is a unique subtype of astrocytes related to HIV-\nassociated pain is rather questionable because those cells may not be a separate\nlineage (as the title suggests) but rather responsive cells to this type of stimuli,\nwhich makes the title misleading. More data is needed to prove the claim that this\nis a lineage-specific population.\nreviewer on the potential risk of being overinterpreting the available data on that HIPA\nwas derived from a novel cell lineage. We have made the following revisions to improve\naccuracy of interpretation: 1). We deleted the original Fig. 7 (the model) in the previous\nmanuscript, 2) We revised the title and the whole MS to emphasize HIPAs as a subtype\nof reactive astrocytes, 3) when discussing the possibility of HIPA being derived from\nependymal cells in DISCUSSION, we clearly stated alternative possibilities and the need\nof direct evidence for validation of our idea (page 11, paragraph 2). An author of this paper\n(AD, a PhD student in the Tang lab) is continuing on this line of investigation to directly\ntest the ependymal hypothesis of HIPA origin; her studies are out of the scope of this\npaper and will be reported separately in the future.\n2. It is unclear if these cells are responsive to pain-related signals in general or just\nto HIV-specific signals? To make this generalization, a non-HIV pain-related animal\nmodel would be helpful to determine if this population is relevant to HIV only or\nother pain models/systems. Otherwise, that claim should be reassessed.\nReviewer #3 (Remarks to the Author):\nIn this interesting article by Zheng and colleagues HIV-Pain Associated Astrocytes\n(HIPA) are examined and the authors identify a specific HIPA population that is\n4\nmarked by Galectin 3. The paper starts out using scRNA-seq from the spinal cords\nof HIPA mouse models and identifies striking differences in the astrocyte\nconstituency. Further analysis identifies Galectin 3 as a prospective marker of this\nHIPA population. Functional studies ensue, where they characterize the origin of\nthis population, its possible functional interactions with neurons, and the role\nGalectin 3 in pain responses.\nOverall, the broad idea of this manuscript is quite enticing as the authors claim to\nhave identified a specific subpopulation of astrocytes linked to HIPA and then\nperform a series of functional studies with that population. However, the\nexperiments are very poorly conducted, the data is of very poor quality, and the\nconclusions are specious (at best). Accordingly, this manuscript does not meet the\nstandard for publication in Nature Communications. Below are a series of specific\ncomments and suggestions that I hope the authors will take under advisement.\n1. The images are very poor quality—particularly figures 3, 4, and 6. Some of these\nimages are out of focus and figures 3-4 have no quantification.\nreviewer and revised DISCUSSION to acknowledge that the engulfment findings need to\nbe confirmed in the future with a second approach, such as the viral labeling approach\ndeveloped by Dr. Chung (p10, paragraph2).\n4. The claim that the Galectin 3 astrocytes are emanating from the central canal is\nalso interesting, but it not at all proven. The images in figure 3 are like the opening\nargument—but to prove this hypothesis, they need to lineage trace cells\nspecifically from the central canal. This would require a Galectin 3-CreER line or\nsome other line that maker from the central canal. Accordingly, another model that\nexplains this phenomenon is that Galectin 3 is simply turned on by astrocytes in\nthe parenchyma.\nResponse. We are grateful for the thoughtful and constructive comment, which is well\ntaken. We consider this comment is like Reviewer #2’s comment #1 and have responded\nthere.\n5. The Galectin-KO studies are interesting----however they are lacking the Galectin-\nKO control (without the gp120Tg). Moreover, the phenotype in Von Frey is very\nweak and is almost non-existent. Furthermore, this experiment is really\nunderpowered (some only have three mice). Finally, the authors should conduct a\ncomprehensive battery of pain-related behaviors. Given the mild effect with Von\nFrey, I would guess that the Galectin-KO; gp120Tg probably didn’t have any other\npain-related phenotypes.\nreviewer that the mechanical pain hypersensitivity manifested by gp120Tg mice was not\nvery drastic. Compared with wild-type mice, gp120Tg mice had a 38.6% drop in mean\nwithdrawal threshold. Nevertheless, the difference reached statistical significance and\nwas repeatable by different team members under blinded conditions.\nFor thermal pain measured by Hargreave testing (Supplementary Figure 9), gp120Tg\nmice also manifested hypersensitivity and this thermal pain hypersensitivity was\nsignificantly attenuated by global Gal3 KO. However, heterozygous KO of astrocytic Gal3\nin the gp120Tg mice did not significantly attenuate thermal pain. We revised RESULTS\nto report the new data (page 7, paragraph 3).\n6. The whole A1/A2 nomenclature is no longer valid and should be removed from\nthe introduction and the paper entirely. See this review (Escartin, et at. Nature\nNeuroscience 2021), as nearly the entire astrocyte field has come to an agreement\nabout this.\nReviewer #2). In fact, we found that HIPAs expressed both ‘A1’ and ‘A2’ markers,\nindicating that HIPAs cannot simply be classified as either A1 or A2 subtypes according\nto the binary scheme of astrocytic subtypes. We considered this finding in revised\nDISCUSSION (page 11, paragraph 3).\nReferences\n1. Trias, E., Barbeito, L. & Yamanaka, K. Phenotypic heterogeneity of astrocytes in motor neuron\ndisease. Clin Exp Neuroimmunol 9, 225-234 (2018).\n2. Komine, O., et al. Innate immune adaptor TRIF deficiency accelerates disease progression of ALS\nmice with accumulation of aberrantly activated astrocytes. Cell Death Differ 25, 2130-2146 (2018).\n3. Liddelow, S.A., et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature\n541, 481-487 (2017).\n4. Addis, D.R., DeBerry, J.J. & Aggarwal, S. Chronic Pain in HIV. Mol Pain 16, 1744806920927276\n(2020).\n5. Yuan, S.B., et al. Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.\nAnn Neurol 75, 837-850 (2014).\n8\nPoint-to-Point Responses\nReviewer #1 (Remarks to the Author):\nThe authors have significantly improved the manuscript by testing the CKO mice. They\nfound that heterozygous CKO of Gal3 significantly attenuated the expression of\nmechanical hypersensitivity of gp120Tg mice (Fig. 7a). They also addressed the issue\nof A1/A2 astrocytes raised by the third reviewer. Overall, this study is a significant\ncontribution to the field of glial biology and pain.\nReviewer #3 (Remarks to the Author):\nThe authors have taken time and great effort to revise their manuscript, however there\nare still several flaws with this study.\n1) While I appreciate the high resolution imaging that suggests that these HIV\nassociated astrocytes are phagocytic, in the absence of functional studies\ndemonstrating this, I remain unconvinced. The comparison between Wt and gp120\nmutant is not convincing, either some sort of in vitro study or viral tool or astrocyte-\nspecific Gal3-KO or KO of phagocytic receptors in Gal3+ astrocytes is very much\nneeded here. Otherwise, it's just a correlation and there is no definitive proof of\nphagocytosis."}, "author_response": ""}]

Decision letter