Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation-an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca²⁺ ] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.

We studied mice with or without heterozygous deletion of the Casr in the parathyroid gland (PTG) [(PTG) CaSR(+/-)] to delineate effects of age and sex on manifestations of hyperparathyroidism (HPT). In control mice, aging induced a left-shift in the Ca(2+) /parathyroid hormone (PTH) set point accompanied by increased PTG CaSR expression along with lowered serum Ca(2+) and mildly increased PTH levels, suggesting adaptive responses of PTGs to aging-induced changes in mineral homeostasis. The aging effects on Ca(2+) /PTH set point and CaSR expression were significantly blunted in (PTG) CaSR(+/-) mice, who showed instead progressively elevated PTH levels with age, especially in 12-month-old females. These 12-month-old knockout mice demonstrated resistance to their high PTH levels in that serum 1,25-dihydroxyvitamin D (1,25-D) levels and RNA expression of renal Cyp27b1 and expression of genes involved in Ca(2+) transport in kidney and intestine were unresponsive to the rising PTH levels. Such changes may promote negative Ca(2+) balance, which further exacerbate the HPT. Skeletal responses to HPT were age-, sex-, and site-dependent. In control mice of either sex, trabecular bone in the distal femur decreased whereas cortical bone in the tibiofibular junction increased with age. In male (PTG) CaSR(+/-) mice, anabolic actions of the elevated PTH levels seemed to protect against trabecular bone loss at ≥ 3 months of age at the expense of cortical bone loss. In contrast, HPT produced catabolic effects on trabecular bone and anabolic effects on cortical bone in 3-month-old females; but these effects reversed by 12 months, preserving trabecular bone in aging mice. We demonstrate that the CaSR plays a central role in the adaptive responses of parathyroid function to age-induced changes in mineral metabolism and in target organ responses to calciotropic hormones. Restraining the ability of the PTG to upregulate CaSRs by heterozygous gene deletion contributes to biochemical and skeletal manifestations of HPT, especially in aging females.

Objective

While latently HIV-infected cells have been described in the blood, it is unclear whether a similar inducible reservoir exists in the gut, where most HIV-infected cells reside. Tissue-specific environments may contribute to differences in the mechanisms that govern latent HIV infection and amenability to reactivation. We sought to determine whether HIV-infected cells from the blood and gut differ in their responses to T-cell activation and mechanistically distinct latency reversing agents (LRAs).

Design

Cross sectional study using samples from HIV-infected individuals (n = 11).

Methods

Matched peripheral blood mononuclear cells (PBMC) and dissociated total cells from rectum ± ileum were treated ex vivo for 24 h with anti-CD3/CD28 or LRAs in the presence of antiretrovirals. HIV DNA and 'read-through', initiated, 5' elongated, completed, and multiply-spliced HIV transcripts were quantified using droplet digital PCR.

Results

T-cell activation increased levels of all HIV transcripts in PBMC and gut cells, and was the only treatment that increased multiply-spliced HIV RNA. Disulfiram increased initiated HIV transcripts in PBMC but not gut cells, while ingenol mebutate increased HIV transcription more in gut cells. Romidepsin increased HIV transcription in PBMC and gut cells, but the increase in transcription initiation was greater in PBMC.

Conclusion

The gut harbors HIV-infected cells in a latent-like state that can be reversed by T-cell activation involving CD3/CD28 signaling. Histone deacetylation and protein kinase B may contribute less to HIV transcriptional initiation in the gut, whereas protein kinase C may contribute more. New LRAs or combinations are needed to induce multiply-spliced HIV and should be tested on both blood and gut.

The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi⁺ RRE⁺), 3' defective (Psi⁺ RRE^-), and 5' defective (Psi^- RRE⁺) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4⁺ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/10⁶ cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/10⁶ cells; 5.6%) or intact (0.6 copies/10⁶ cells; <1%) HIV RNA. Likewise, the frequency of CD4⁺ T cells with 3' defective HIV RNA was greater than the frequency with 5' defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 3' defective RNA over 5' defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. IMPORTANCE We developed a new assay that can distinguish and quantify intact (potentially infectious) as well as defective HIV RNA. In ART-treated individuals, we found that the vast majority of all HIV RNA is defective at the 3' end, possibly due to incomplete transcriptional processivity. Only a very small percentage of all HIV RNA is intact, and very few total or intact proviruses transcribe intact HIV RNA. Though rare, this intact HIV RNA is tremendously important because it is necessary to serve as the genome of infectious virions that allow transmission and spread, including rebound after stopping ART. Moreover, intact viral RNA may contribute disproportionately to the immune activation, inflammation, and organ damage observed with untreated and treated HIV infection. The intact viral RNA assay can be applied to many future studies aimed at better understanding HIV pathogenesis and barriers to HIV cure.

Although there have been great advancements in the field of HIV treatment and prevention, there is no cure. There are two types of HIV: HIV-1 and HIV-2. In addition to genetic differences between the two types of HIV, HIV-2 infection causes a slower disease progression, and the rate of new HIV-2 infections has dramatically decreased since 2003. Like HIV-1, HIV-2 is capable of establishing latent infection in CD4+ T cells, thereby allowing the virus to evade viral cytopathic effects and detection by the immune system. The mechanisms underlying HIV latency are not fully understood, rendering this a significant barrier to development of a cure. Using RT-ddPCR, we previously demonstrated that latent infection with HIV-1 may be due to blocks to HIV transcriptional elongation, distal transcription/polyadenylation, and multiple splicing. In this study, we describe the development of seven highly-specific RT-ddPCR assays for HIV-2 that can be applied to the study of HIV-2 infections and latency. We designed and validated seven assays targeting different HIV-2 RNA regions along the genome that can be used to measure the degree of progression through different blocks to HIV-2 transcription and splicing. Given that HIV-2 is vastly understudied relative to HIV-1 and that it can be considered a model of a less virulent infection, application of these assays to studies of HIV-2 latency may inform new therapies for HIV-2, HIV-1, and other retroviruses.

Latently-infected CD4+ T cells are widely considered to be the major barrier to a cure for HIV. Much of our understanding of HIV latency comes from latency models and blood cells, but most HIV-infected cells reside in lymphoid tissues such as the gut. We hypothesized that tissue-specific environments may impact the mechanisms that govern HIV expression. To assess the degree to which different mechanisms inhibit HIV transcription in the gut and blood, we quantified HIV transcripts suggestive of transcriptional interference (U3-U5; "Read-through"), initiation (TAR), 5' elongation (R-U5-pre-Gag; "Long LTR"), distal transcription (Nef), completion (U3-polyA; "PolyA"), and multiple splicing (Tat-Rev) in matched peripheral blood mononuclear cells (PBMCs) and rectal biopsies, and matched FACS-sorted CD4+ T cells from blood and rectum, from two cohorts of ART-suppressed individuals. Like the PBMCs, rectal biopsies showed low levels of read-through transcripts (median = 23 copies/106 cells) and a gradient of total (679)>elongated(75)>Nef(16)>polyadenylated (11)>multiply-spliced HIV RNAs(<1) [p<0.05 for all], demonstrating blocks to HIV transcriptional elongation, completion, and splicing. Rectal CD4+ T cells showed a similar gradient of total>polyadenylated>multiply-spliced transcripts, but the ratio of total to elongated transcripts was 6-fold lower than in blood CD4+ T cells (P = 0.016), suggesting less of a block to HIV transcriptional elongation in rectal CD4+ T cells. Levels of total transcripts per provirus were significantly lower in rectal biopsies compared to PBMCs (median 3.5 vs. 15.4; P = 0.008) and in sorted CD4+ T cells from rectum compared to blood (median 2.7 vs. 31.8; P = 0.016). The lower levels of HIV transcriptional initiation and of most HIV transcripts per provirus in the rectum suggest that this site may be enriched for latently-infected cells, cells in which latency is maintained by different mechanisms, or cells in a "deeper" state of latency. These are important considerations for designing therapies that aim to disrupt HIV latency in all tissue compartments.

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor β (TGF-β), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-β signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103⁺ and CD103^- CD4 T cells from the blood and rectum of HIV-negative (HIV^-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV⁺) individuals. Like gut CD4⁺ T cells, circulating CD103⁺ cells harbored more HIV DNA than did CD103^- cells but transcribed less HIV RNA per provirus. Circulating CD103⁺ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103^- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103⁺ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103⁺ CD4⁺ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103⁺ T cells share a cellular transcriptome that more closely resembles that of CD4⁺ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4⁺ or circulating CD103⁺ T cells from circulating CD103^- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.