key: cord-0951702-4lh2swjd
authors: Schmidt, Sarah; Schenkova, Kristina; Adam, Tarek; Erikson, Elina; Lehmann‐Koch, Judith; Sertel, Serkan; Verhasselt, Bruno; Fackler, Oliver T.; Lasitschka, Felix; Keppler, Oliver T.
title: SAMHD1's protein expression profile in humans
date: 2015-02-02
journal: J Leukoc Biol
DOI: 10.1189/jlb.4hi0714-338rr
sha: 5f79344199348697e8f7eb8fa5d9f09235e6685b
doc_id: 951702
cord_uid: 4lh2swjd

The deoxynucleoside triphosphate triphosphohydrolase and 3′ → 5′ exonuclease SAMHD1 restricts HIV‐1 infection in noncycling hematopoietic cells in vitro, and SAMHD1 mutations are associated with AGS. Little is known about the in vivo expression and functional regulation of this cellular factor. Here, we first assessed the SAMHD1 protein expression profile on a microarray of 25 human tissues from >210 donors and in purified primary cell populations. In vivo, SAMHD1 was expressed in the majority of nucleated cells of hematopoietic origin, including tissue‐resident macrophages, DCs, pDCs, all developmental stages of thymic T cells, monocytes, NK cells, as well as at lower levels in B cells. Of note, SAMHD1 was abundantly expressed in HIV target cells residing in the anogenital mucosa, providing a basis for its evaluation as a cellular factor that may impact the efficiency of HIV transmission. Next, we examined the effect of the activation status and proinflammatory cytokine treatment of cells on expression and phosphorylation of SAMHD1. Activated, HIV‐susceptible CD4(+) T cells carried pSAMHD1(T592), whereas resting CD4(+) T cells and macrophages expressed the unphosphorylated protein with HIV‐restrictive activity. Surprisingly, stimulation of these primary cells with IFN‐α, IFN‐γ, IL‐4, IL‐6, IL‐12, IL‐18, IL‐27, or TNF‐α affected neither SAMHD1 expression levels nor threonine 592 phosphorylation. Only IL‐1β moderately down‐regulated SAMHD1 in activated CD4(+) T cells. Taken together, this study establishes the first cross‐sectional protein expression profile of SAMHD1 in human tissues and provides insight into its cell cycle‐dependent phosphorylation and unresponsiveness to multiple proinflammatory cytokines.

Mutations within the SAMHD1 gene lead to a rare genetic disorder, the AGS, a primary immunodeficiency disorder that resembles an immune system's response pattern to certain chronic viral infections, displaying a massive production of the antiviral cytokine IFN-a and a detrimental activation of the immune system [1] . The deficiency in SAMHD1 expression results in a complex disease characterized by hepatosplenomegaly, a chronic cerebrospinal fluid lymphocytosis, and an early-onset encephalopathy that leads to severe intellectual and physical handicap [2] . The potential involvement of SAMHD1 in innate immunity had been suggested initially by studies of its mouse ortholog Mg11, which is IFN inducible in macrophages and DCs [3] . Subsequent studies showed an increased SAMHD1 expression upon stimulatory DNA treatment of macrophages [4, 5] and its up-regulation in the context of viral infections [6] [7] [8] . Whereas the SAM domain mediates proteinprotein interactions, the HD domain possesses dNTP triphosphohydrolase activity [9, 10] , through which SAMHD1 hydrolyzes dNTPs to deoxynucleosides. SAMHD1 was identified as a potent restriction factor for HIV [11, 12] , other nonhuman retroviruses [13] , and herpesviruses, including HSV-1 [14, 15] . For HIV, the current mode-of-action model suggests that SAMHD1, through its enzymatic function as a dNTP triphosphohydrolase, diminishes intracellular dNTP pools in noncycling cells, decreasing their concentrations below a threshold required for efficient HIV-1 cDNA synthesis by the viral RT [13, [16] [17] [18] . Furthermore, a metal-dependent 39 → 59 exonuclease activity of SAMHD1 against ssDNAs and ssRNAs was demonstrated in vitro [19] . The RNAse activity was suggested recently to play a pivotal role for the HIV-restrictive activity of SAMHD1 by directly degrading the HIV-1 RNA of incoming particles [20] .

A few studies in cultured HIV target cells have documented SAMHD1 expression in monocytes, MDMs, MDDCs, pDCs, and CD4 + T cells [12, 16, [21] [22] [23] [24] . However, sites of SAMHD1 expression in vivo have not been systematically defined.

The primary aim of our study was thus to establish the in vivo expression profile of SAMHD1 in human tissues, with a focus on defining cells that harbor this nucleotide metabolismmodulating enzyme and that are also target cells of HIV-1 and HSV. In the context of its HIV-restrictive activity, we also sought to assess the effect of T cell activation and stimulation with a number of proinflammatory cytokines on the overall expression level, as well as pSAMHD1(T592). In addition, we aimed at identifying SAMHD1-expressing cells that may play a role in AGS pathophysiology.

All paraffinized tissue specimens were provided by the tissue bank of the National Center for Tumor Diseases (Heidelberg, Germany) and their use approved by Heidelberg University's Ethics Committee (Germany; Approval No. 206/2005) . Based on these specimens, an in-house, whole-body TMA containing a total of 468 paraffinized tissue sections from 25 different healthy organs from over 210 donors in total was assembled [25] . This TMA consists of a total of 5 coverslips, each containing 90-100 round sections (diameter of 2 mm) from paraffinized tissue blocks (see Fig. 1A and B, for examples). In more detail, histopathological tissue blocks, without evidence of tumor and/or inflammation, were selected from donors that had undergone surgery for non-neoplastic reasons, i.e. tonsillitis, diverticulosis, and phimosis, or for neoplastic reasons, i.e. colorectal carcinoma or lung carcinoma. Other tissues, i.e., heart and brain, were derived from autopsies. In any case, the tissues used for the construction of the TMA were tumor and inflammation free, as assessed by histopathology. Donors were 60% men/boys and 40% women/girls, and ages were between 5 and 82 yr. Underlying conditions other than the indication for surgery have not been evaluated. Healthy tissues represented on the TMA analyses were salivary gland (n = 15), thyroid (n = 16), thymus (n = 10), lung (n = 20), heart (n = 21), esophagus (n = 21), stomach (n = 20), small intestine (n = 19), large intestine (n = 24), liver (n = 18), gallbladder (n = 14), spleen (n = 27), pancreas (n = 7), kidney (n = 24), adrenal gland (n = 5), bladder (n = 7), prostate (n = 25), testis (n = 22), uterus (n = 5), skin (n = 5), fat (n = 14), skeletal muscle (n = 11), ovary (n = 7), tonsil (n = 8), and appendix (n = 8). After immunohistochemical staining for SAMHD1, TMA slides were scanned for image acquisition by use of the Aperio ScanScope CS (Leica Microsystems, Buffalo Grove, IL, USA), analyzed with Aperio ImageScope software (Leica Microsystems), and rated, in principle, based on the immunoreactive scoring system of Allred et al. [26] and as recently reported [25] . Each section was assigned 2 scores: 1 score for the fraction of cells that stained positive (0, ,1% positive cells; 1, 1-10% positive cells; 2, 11-33% positive cells; 3, 34-66% positive cells; 4, .66% positive cells) and another score for the intensity of staining (0, no staining; 1, weak staining; 2, intermediate staining; 3, strong staining). The overall amount of SAMHD1 was then calculated as the sum of the scores for the portion of positive, tissuespecific cells and for staining intensity. A total score of 7 was categorized as high staining, scores of 5 and 6 as medium staining, and scores of 2-4 as low staining (see Fig. 1C ).

Antigen retrieval and immunoenzyme staining were performed as described [25] . Preimmune rabbit IgG served as a negative control. Immunoenzyme stainings of SAMHD1 were performed on 2 mm paraffin sections of formalinfixed tissues by use of standard avidin-biotin antialkaline phosphatase techniques (Vectastain; Vector Laboratories, Burlingame, CA, USA; Super Sensitive Link-Label IHC Detection System; BioGenex, Fremont, CA, USA). Slides were viewed with an Olympus BX45 microscope (Olympus, Center Valley, PA, USA), and pictures were taken with an Olympus U-CMAD3 F-View camera (Olympus).

Double-immunofluorescence stainings were performed by use of pairwise combinations of primary antibodies. As a secondary reagent, biotinylated goat anti-mouse antibodies were used for 30 min at room temperature, followed by Cy3-conjugated streptavidin antibodies. The SAMHD1 rabbit primary reagent was directly detected by use of an anti-rabbit antibody coupled to Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA). Pictures were taken at an Olympus IX81 microscope with an Olympus U-CMAD3 F-View camera and imported into cell^M software or with a Nikon Eclipse Ti microscope (Nikon Instruments, Melville, NY, USA) by use of an Orca-Flash 2.8 camera (Hamamatsu, Hamamatsu City, Japan) and imported into NIS elements Basic Research software (Nikon Instruments). All primary and secondary antibodies used in this study are listed in Supplemental Table 1 .

Cells and organ biopsies were resuspended directly in SDS lysis buffer, containing 4% SDS, or lysed in NP-40 buffer (1% NP-40, 150 mM NaCl, 40 mM Tris, pH 8) for 30 min on ice. Proteins were applied on a 10% or 12.5% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. Blocked membranes were consecutively probed with rabbit (Proteintech Europe, Manchester, United Kingdom) or mouse (Abcam, Cambridge, United Kingdom) polyclonal anti-SAMHD1 antiserum for detection of pan-SAMHD1 expression and an anti-MAPK and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-MLC (Sigma-Aldrich, St. Louis, MO, USA) antibody (Supplemental Table 1 ). For detection of pSAMHD1 (T592), rabbits were immunized with peptide IAPLI(pT)PQKKE, and immunoreactive sera were affinity purified [22] . Cells were lysed and applied on a 4-20% gradient SDS-PAGE. After transfer, polyvinylidene difluoride membranes were probed consecutively with primary antibodies/antisera for detection of the indicated proteins. Secondary antibodies were conjugated to HRP for ECL-based detection.

For fixation and antibody staining, PBMCs, freshly isolated tonsil cells, or thymocytes were fixed for 10 min at room temperature with BD Cytofix Fixation Buffer (BD Biosciences, San Jose, CA, USA) and permeabilized in BD Phosflow Perm Buffer III (BD Biosciences) for 2 min (PBMCs, tonsil) or 30 min (thymocytes) at 4°C in the dark. After 2 washes (PBS/1% FCS/0.09% sodium azide), cells were stained with the rabbit anti-pan-SAMHD1 antiserum, followed by Alexa 660-conjugated goat anti-rabbit antibodies and the respective directly labeled markers. A control rabbit antiserum as well as SAMHD1-negative Jurkat-TAg cells were always included as informative references (see also Fig. 2A) . A FACSCalibur flow cytometer and BD CellQuest Pro 4.0.2 software (BD PharMingen, San Diego, CA, USA) were used for analyses. For flow cytometric analysis of thymocytes, cells were first stained in staining buffer (PBS, 1% BSA, and 0.09% sodium azide) for surface markers, followed by fixation and permeabilization for pan-SAMHD1 staining, as described above. Flow cytometric analysis was carried out on an LSR II cytometer by use of FACSDiva software (both BD Biosciences). The live cell population was gated based on forwardand side-scatter.

Human cell lines THP-1 and Jurkat-TAg were cultivated under standard conditions, as reported previously [27, 28] . PBMCs from healthy blood donors [approved by Frankfurt University's Ethics Committee (Approval No. 329/10), Germany] were purified by Ficoll-Hypaque gradient centrifugation, in (continued from previous page) histidine/aspartic acid domain-containing protein 1 at threonine 592, SAMHD1 = sterile alpha motif and histidine/aspartic acid domain-containing protein 1, TAg = SV40 large T antigen, TMA = tissue microarray principle, as reported [29] Subsequently, cells were isolated by magnetic labeling of the respective lineage marker (MicroBeads; Miltenyi Biotec, San Diego, CA, USA), followed by magnetic separation (either positive or negative) via an AutoMACS (Miltenyi Biotec).

The following cell purities were obtained for the positively selected cells (see Fig. 3C ): CD4 + T cells (99%), CD14 + monocytes (90-92%), CD20 + CD19selected B cells (66-72%), CD8 + T cells (97%), CD56 + NK cells (97%), CD11c + MDDCs (92%). To obtain MDM, negatively separated CD14 + monocytes were differentiated in DMEM containing 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% human AB serum for 10 days. CD4 + T cells were isolated with the RosetteSep Human CD4 + T Cells Enrichment Cocktail (Stemcell Technologies, Vancouver, BC, Canada) and cultivated in the absence (resting) or presence (activated) of PHA-P (5 mg/ml; Sigma-Aldrich) and IL-2 (100 IU/ml; Biomol, Enzo Life Sciences, Farmingdale, NY, USA) or anti-CD3/anti-CD28 antibodies (each 1 mg/ml; BD Biosciences) and IL-2 (100 IU/ml; Biomol, Enzo Life Sciences), as described previously [21] .

Child thymus tissue, removed during cardiac surgery, was obtained and used following the guidelines of the Medical Ethical Commission of Ghent University Hospital (Belgium; B670201215078). Informed consent was obtained and samples used according to the Declaration of Helsinki. Thymus suspension was obtained after mechanical homogenization of the tissue, and mononuclear cells were isolated by density-layer centrifugation (Lymphoprep; Axis-Shield, Oslo, Norway). Tonsil tissue was removed during routine tonsillectomy from HIV-, hepatitis B virus-, and hepatitis C virusnegative patients with informed consent. The use of anonymous surgical waste was approved by Heidelberg University's Ethics Committee (Approval No. 077/2005). Fresh tonsil cultures were prepared by collagenase digestion (collagenase type I; Worthington Biochemical, Lakewood, NJ, USA) at 37°C for 15 min, followed by mechanical homogenization and PBS washing. The resulting single-cell suspension was directly stained and applied to flow cytometric analysis.

For cytokine treatment, MDM or CD4 + T cells were incubated with IFN-a (Roche, Basel, Switzerland); IFN-g, IL-4, IL-12, IL-27, and TNF-a (PeproTech, Rocky Hill, NJ, USA); and IL-6 and IL-1b (R&D Systems) or IL-18 (MBL International, Woburn, MA, USA) at concentrations ranging between 50 and 10.000 IU/ml (or ng/ml) as indicated. After 24 h or 48 h, cells were lysed and applied to immunoblot analysis or fixed and stained for intracellular SAMHD1 and subsequent flow cytometric analysis.

To assess statistical significance, the unpaired Student's t-test was used. P . 0.05 was considered not significant.

A cross-sectional SAMHD1 expression profiling was performed on a TMA containing 468 individual sections derived from 25 paraffinized nontransformed tissues from over 210 patients. First, we surveyed SAMHD1 protein expression on the TMA, based on a combined proportion and intensity scoring system, in principle, as reported [25, 26] . SAMHD1-expressing cells were detected in all organs analyzed ( Fig. 1 and Supplemental Fig. 1A and B), with expression scores varying considerably among tissues: the scores of small intestine, appendix, spleen, esophagus, tonsil, ovary, pancreas, and thymus were highest (.80% medium or highexpression score). Generally low or negative expression scores were determined for fatty tissue, uterus, liver, and muscle ( Fig. 1 and Supplemental Fig. 1A and B) .

In a complementary approach, strong SAMHD1 expression in homogenates of human tissue samples was confirmed by immunoblot analyses of snap-frozen tissue samples of gallbladder, tonsil, spleen, and pancreas ( Fig. 2A) . The SAMHD1-negative human T cell line Jurkat-TAg served as a specificity control. Besides the major protein band at 72 kDa, seen in all tissues and reported previously in cell lines [11, 12] , additional, prominent, lower molecular weight bands were noted, particularly in a spleen lysate ( Fig. 2A) and some of the purified cell populations from peripheral blood (Fig. 3C) , which may be a result of splice variants, post-translational modifications, or partial proteolytic cleavage. Collectively, although expression levels were variable, SAMHD1 was detected in all organs and tissues analyzed.

Recently, SAMHD1, expressed in cultured monocytes, MDDCs, MDMs, and resting CD4 + T cells, was demonstrated to function as a restriction factor for HIV-1 infection [11, 21, 23, 27, 30] . Given the potential role of SAMHD1 in limiting transmission of HIV-1, we explored its expression in anogenital mucosa and gut-associated lymphoid tissue. Immunohistochemical staining of vagina, foreskin, anal mucosa and rectum tissue sections revealed abundant, constitutive expression of the restriction factor in infiltrating and resident-nucleated hematopoietic cells in mucosa and submucosa of these transmission site tissues (Fig. 2B-D 

By histomorphological criteria, we identified SAMHD1 expression in a number of specialized or organ-specific cells in the TMA: pneumocytes in the lung, exocrine secretory ducts in salivary glands and pancreas, epithelium of the gallbladder and esophagus (stratum basale), endometrium of the uterus, stroma of the ovary, seminiferous tubules of the testis, cells in the adrenal gland with a stronger expression in the zona glomerulosa and zona reticularis compared with the zona fasciculata, keratinocytes in the basal layer of the skin, and gastric glands ( Fig. 1A and B and Supplemental Fig. 1A and B ). In addition, neurocytes of peripheral nerves in vagina stained highly positive for SAMHD1 (Fig. 2B) . Furthermore, coimmunofluorescence analyses of SAMHD1 (primarily nuclear staining) and the endothelial marker CD31 (membranous-cytoplasmic staining) revealed marked coexpression of the triphosphohydrolase/exonuclease in vascular endothelial cells in brain, tonsil, vagina, and rectum (Fig. 2D) , as well as in liver and pancreas (data not shown).

Next, we performed a systematic, in-depth analysis of SAMHD1 expression in primary cells of hematopoietic origin. Based on flow cytometric codetection with lineage-specific markers, histomorphological criteria, costainings in tissue sections, and immunoblotting of purified cell populations, SAMHD1 expression could be demonstrated in all hematopoietic lineages. In tissue, SAMHD1 expression was documented in CD68 + macrophages ( Fig. 3A and Supplemental Fig. 3) , DC-SIGN + DC, BDCA2 + pDC (Fig. 3A) , and thymic and tonsillar CD4 + and CD8 + T cells (Figs. 3 and 4) . Isolated primary cell populations showed expression of SAMHD1 in CD14 + monocytes ( Fig. 3B and C) , MDM (Fig. 3C) , MDDC (Fig. 3C) , CD56 + NK cells (Fig. 3C) , and peripheral CD4 + and CD8 + T cells (Figs. 3B and C and 5A and B). In CD19 + and CD22 + B cells, as well as CD138 + plasma cells, low-level expression ( In the thymus, a lymphoid organ, particularly relevant for HIV pathogenesis [31, 32] , SAMHD1, was expressed in various lymphoid and nonlymphoid cell types ( Fig. 4 and Supplemental Fig. 5 ). The triphosphohydrolase/exonuclease was present during all stages of thymocyte development in 66-86% of cells starting from CD34 + CD1 2 thymic immigrants up to CD4 + CD8 + and single-positive thymocytes ( Fig. 4C and Supplemental Fig. 5 ). There was a slight trend toward increased SAMHD1 expression with increasing thymocyte maturation. In addition, SAMHD1 was found in CD68 + macrophages in the thymic cortex and in mature and immature medullary epithelial cells ( Fig. 4A and B, and data not shown) .

Based on the identification of SAMHD1 expression in a large number of nucleated cell types of the hematopoietic lineage, the high SAMHD1 staining scores in the TMA in all lymphatic organs (e.g., thymus, spleen, tonsil; Fig. 1 ) and intestinal-tract samples with associated lymphatic tissue (e.g., small intestine, appendix, colon; Figs. 1 and 2C), as well as low to medium scores in organs with a significant fraction of resident, transient, or infiltrating mononuclear hematopoietic cells (e.g., liver, thymus, gallbladder, kidney), are readily explained. Thus, this cross-sectional analysis reveals that constitutive SAMHD1 expression can be found in the majority of nucleated cells of hematopoietic origin.

We next wanted to explore whether major proinflammatory cytokines can affect overall expression levels of SAMHD1. To this end, PBMC-derived CD4 + T cells and tonsil-derived lymphoid aggregate cultures (Fig. 5) , as well as MDMs (Fig. 6) , were cultivated in the presence or absence of different concentrations of human IFN-a, IFN-g, IL-4, IL-6, IL-12, IL-18, IL-27, IL-1b, or TNF-a, alone or in combination, for 24 or 48 h. Subsequent analyses by immunoblotting (Figs. 5A and B and 6 ) and intracellular flow cytometry (Fig. 5C ) revealed no significant cytokine-mediated alterations of SAMHD1 expression levels. As a notable exception, the stimulation of activated CD4 + T cells by IL-1b induced a moderate decrease of SAMHD1 expression (Fig. 5B) . As controls of cytokine activity, treatment with IFN-a or IFN-g stimulated MxA or IFI16 expression, respectively, in these HIV target cells (Figs. 5A and 6 ). We next explored whether the activation stimulus affects SAMHD1 expression levels in CD4 + T cells. To this end, CD4 + T cells were exposed to PHA-P/IL-2 or anti-CD3/anti-CD28 antibodies, Figure 1 . SAMHD1 is expressed in the majority of human tissues. (A and B) Images of immunohistochemical stainings of TMA slides. Stainings were performed with a specific polyclonal antipan-SAMHD1 antibody [21] (right) or a rabbit control serum (left), followed by biotinylated donkey anti-rabbit secondary antibodies. Subsequently, sections were exposed to the avidincontaining ABC-AP kit, followed by substrate development with New Fuchsin. Nuclei were counterstained with hematoxylin. (A) Selected tissues, staining positive, mainly for resident or infiltrating mononuclear hematopoietic cells. (B) Selected tissues, displaying organ-specific or specialized SAMHD1-positive cells. (C) Rating of SAMHD1 expression in human tissues. The expression of SAMHD1 was quantified with a proportion and intensity scoring system [25, 26] . Histogram bars depict the percentage of cases with high (black), medium (gray), or low (open) expression ratings. The percentage of samples with negative ratings is not shown. together with IL-2 (Fig. 5B ). Neither of these T cell activation protocols significantly affected SAMHD1 levels, as reported previously [21, 24] .

As pSAMHD1(T592) has been reported to correlate with a loss of its HIV-restrictive capacity [23, 33] , we also tested whether cytokine stimulation altered the phosphorylation status of SAMHD1 in these primary target cells. To this end, we generated an affinity-purified, T592 phosphosite-specific polyclonal rabbit antiserum. Nitrocellulose membranes with separated lysates from cytokine-treated cells were reprobed with this antiserum. pSAMHD1(T592) was detectable in primary CD4 + T cells only following T cell activation but not in quiescent cells from the same donor ( Fig. 5A and B) . Remarkably, pSAMHD1 (T592) expression was unaltered by cytokine treatment in resting and activated CD4 + T cells (Fig. 5A and B) . In contrast to cycling CD4 + T cells, pSAMHD1(T592) was undetectable in differentiated MDM, and also for these myeloid cells, neither IFN-a, IFN-g, IL-12/IL-18, nor TNF-a stimulation had an impact on this status (Fig. 6) . Collectively, neither T cell activation nor the addition of major proinflammatory cytokines, with the notable exception of IL-1b, modulated SAMHD1's expression levels or phosphorylation at threonine 592 in primary lymphoid and myeloid HIV target cells.

Our cross-sectional in vivo profiling of the dNTP triphosphohydrolase/39 → 59 exonuclease SAMHD1 identified its widespread expression in nontransformed tissues, including a number of highly specialized and organ-specific cell types throughout the human body, as well as in the majority of nucleated cell types of hematopoietic origin. Our whole-body TMA expression approach allowed us to examine cellular in vivo targets of pathogenic viruses that have been shown to be inhibited by the nucleotide-modulating antiviral factor SAMHD1 in tissue culture. For cells that can, in principle, be infected by HIV-1 in vivo, SAMHD1 in situ expression was demonstrated in monocytes, macrophages, DCs, pDCs, and mature CD4 + T cells, as well as in all developmental stages of thymic T cells. Thus, virtually all major HIV target cells in an infected individual potentially carry the SAMHD1 protein. keratinocytes, and neurocytes were found to express SAMHD1, also suggesting that this viral pathogen may frequently encounter the restriction factor in the context of its natural infection. In the lung, alveolar pneumocytes, which express the restriction factors SAMHD1 (present study), as well as CD317/tetherin [25] , are targeted by various respiratory pathogens, including influenza viruses [34] , severe acute respiratory syndrome coronavirus [35] , and respiratory syncytial virus [36] . At least for CD317, influenza viruses seem to have evolved mechanisms of viral antagonism [37, 38] . For these respiratory viruses, it is currently unclear whether SAMHD1 impacts their replication and/or whether they encode a SAMHD1 antagonist similar to the viral protein X proteins of HIV-2 and certain SIV strains [39] .

Giventhe dNTP triphosphohydrolase and 39 → 59 exonuclease activities of SAMHD1 and the impact that these nucleotidemodulating activities could have on cell-cycle progression and cellular proliferation, we wondered whether its in vivo expression pattern was restricted to noncycling cells. This was clearly not the case: SAMHD1 was expressed in cycling, highly proliferative primary cells (e.g., endometrium and activated T cells), as well as noncycling cells (e.g., vascular endothelium, resting T cells, and macrophages). This broad expression pattern suggests that the regulation of the physiologic functions of SAMHD1 may occur by direct modulation of its enzymatic and antiviral activities rather than at the transcriptional/translational level. In line with this notion, recent studies reported that cyclin-dependent kinase 1 mediated phosphorylation of SAMHD1 at residue T592 regulates its capacity to restrict HIV-1 [22, 23] . Interestingly, pSAMHD1(T592) does not seem to regulate the protein's Histogram bars represent the arithmetic mean + SD of the mean fluorescence intensity (MFI) of SAMHD1 expression in the respective thymocyte subsets of 4 donors. Thymocyte differentiation was categorized into the 7 stages indicated: 1) CD34 + CD1 2 , recent thymic immigrants; 2) CD3 + CD4 + CD8 2 , immature, single-positive cells; 3) CD3 2 CD4 + CD8 + , double-positive thymocytes undergoing b-selection; 4) CD3 + CD4 + CD8 + , doublepositive thymocytes that have undergone b-selection; 5) CD3 + CD27 + CD69 + , positively selected thymocytes; 6) CD3 + CD4 2 CD8 + , mature CD8 + thymic T cells; and 7) CD3 + CD4 + CD8 2 , mature CD4 + thymic T cells. The numbers indicated below the histogram represent the percentage of SAMHD1 + cells within the respective subset. catalytic dNTPase activity [23, 33, 40] , suggesting, at least in part, a regulatory uncoupling of its antiviral and enzymatic functions.

It is important to define which cytokines and stimuli affect expression and function of SAMHD1 in primary HIV target cells. Recently, Cribier et al. [33] reported the loss of pSAMHD1 (T592) in activated CD4 + T cells and MDM following exposure to IFN-a. Whereas in our study, pSAMHD1(T592) was also readily detected in CD4 + T cells upon TCR stimulation, in line with other reports [23, 33] , we did not observe a reduction in SAMHD1 phosphorylation by treatment with IFN-a or IFN-g, despite an apparent induction of type I and type II IFN-positive controls MxA and IFI16, respectively. A recent study reported an ;2-fold induction of SAMHD1 expression in MDM by treatment with a combination of IL-12 and IL-18, each at 100 ng/ml [41] . We were neither able to confirm this cytokine-mediated up-regulation of the restriction factor at 100 or 1000 ng/ml nor did we observe changes in the pSAMHD1(T592) expression of cells. Likewise, this cytokine combination neither affected overall levels nor phosphorylation of SAMHD1 in CD4 + T cells. In line with our observation, St Gelais et al. [24] did not observe an increase in SAMHD1 levels in CD4 + T cells or MDDCs following exposure to type I IFN. Of note, IL-1b was the only cytokine that down-regulated SAMHD1 significantly in activated CD4 + T cells. However, it is unclear whether this translates into any change in the HIV susceptibility of cells, as SAMHD1 is not believed to pose a restriction for HIV in proliferating T cells. For assessment of the SAMHD1 status and cytokine responses in MDM, the monocyte isolation and differentiation protocol may be of particular relevance: the M-CSF-based rapid differentiation protocol (3 days) used in 1 study [22] resulted in strong pSAMHD1(T592) expression. In contrast, our results, in agreement with Allouch et al. [42] , showed a low-level or absent pSAMHD1(T592) expression in MDM differentiated with human AB serum for 7-10 days. Besides differentiation regimes, the purity of the initial monocyte preparation from PBMC may also affect this analysis, as residual T cells can become activated during the monocyte differentiation process and thus, contribute to an appreciable pSAMHD1(T592) signal in an immunoblot analysis of bulk cell populations. Collectively, the reported discrepancies of SAMHD1 expression, phosphorylation, and regulation in primary CD4 + T cells and MDM may, in part, stem from differences in methodology and will have to be addressed carefully by additional studies.

The whole-body TMA approach identified constitutive SAMHD1 protein expression in vascular endothelium in a number of tissues, including the CNS, gastrointestinal tract, lymphatic tissues, genital tract, liver, and pancreas. This is of particular interest, as cerebral and cutaneous symptoms in AGS patients have been linked to vasculature [43] . Tubuloreticular inclusions in endothelial cells and cerebral arterial stenoses are found in the context of stroke in these patients. Moreover, half of the patients with AGS have painful, itchy skin lesions [44] that are associated with an inflammation of small blood vessels. Together, one can speculate that under conditions of SAMHD1 deficiency, an inappropriate accumulation of nucleic acids triggers autoimmune responses [1, [45] [46] [47] that underlie the development of the observed inflammatory processes.

The broad expression profile of SAMHD1 may, in part, allow SAMHD1 to act as a ubiquitous guard that protects cells from retroviral insult. Based on the current understanding of its antiviral mode of action, the effectiveness of this cellular barrier depends on the cycling and possibly phosphorylation status of target cells. Taken together, the cross-sectional SAMHD1 expression profile established in the current report will instruct studies into the biologic role of this nucleic acid-modulating enzyme in physiology and infectious diseases. 

Aicardi-Goutieres syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity

Aicardi-Goutières syndrome

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2012) SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates

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SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutières syndrome are highly susceptible to HIV-1 infection

HIV-1 Vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor CD317/tetherin to overcome the virion release restriction

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Thymus involution in the acquired immunodeficiency syndrome

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Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1

Comparison of the pathology caused by H1N1, H5N1, and H3N2 influenza viruses

Pathogenetic mechanisms of severe acute respiratory syndrome

The histopathology of fatal untreated human respiratory syncytial virus infection

Modulation of an ectodomain motif in the influenza A virus neuraminidase alters tetherin sensitivity and results in virus attenuation in vivo

Budding capability of the influenza virus neuraminidase can be modulated by tetherin

New insights into an X-traordinary viral protein

Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1

Restriction of HIV-1 replication in primary macrophages by IL-12 and IL-18 through the upregulation of SAMHD1

Reply to Pauls et al.: p21 is a master regulator of HIV replication in macrophages through dNTP synthesis block

Cerebral arterial stenoses and stroke: novel features of Aicardi-Goutières syndrome caused by the Arg164X mutation in SAMHD1 are associated with altered cytokine expression

Chilblains as a diagnostic sign of aicardi-goutières syndrome

Plasmacytoid dendritic cells and interferon-alpha in Aicardi-Goutières syndrome

Aicardi-Goutières syndrome and systemic lupus erythematosus (SLE) in a 12-year-old boy with SAMHD1 mutations

Nucleic acid metabolism and systemic autoimmunity revisited

KEY WORDS: innate immunity • restriction factor • HIV • HSV

This work was, in part, funded by grants from BioBanking (CRC938/ TP Z2 and DZIF/TI; to F.L.), and Deutsche Forschungsgemeinshaft Grants KE742/5-1 (to O.T.K.) and FA 398/13/1 (to O.T.F.). The authors thank the Robert Koch-Institute for financial support of the National Reference Center for Retroviruses. The authors are grateful for support from Goethe University. The authors thank Hanna-Mari Baldauf for support and Bruno Kyewski for discussion. The authors thank Jutta Scheuerer for technical assistance and Pieter Meuwissen for supervised experimental contributions.

The authors declare no conflict of interest.