Senescent cells promote tissue NAD+ decline during ageing via the activation of CD38+ macrophages

May combine two claims.

When M1 macrophages are compared with M0 and M2, Cd38 mRNA rises roughly 600-fold and Cd157 rises less, whereas Sirt1, Parp1, and Parp2 mRNAs do not rise strongly.

“In M1, relative to M0 and M2, macrophages, the only significantly upregulated NADase-encoding mRNAs were Cd38 (600-fold increase) and, to a lesser degree, its homologue Cd157; Sirt1, Parp1 and Parp2 mRNAs were not highly upregulated in M1 relative to M0 and M2 macrophages (Fig. 1a).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

In M1 macrophages, FK866 lowers glycolytic capacity, and NMN reverses this effect.

“FK866-treated M1 macrophages showed reduced glycolytic capacity, which was rescued by addition of NMN (Fig. 2c)”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

NAD and NADP levels are elevated in M2 but not M1 macrophages.

“We also observed elevated levels of NAD and NADP in M2, but not M1, macrophages (Fig. 1d)”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

In M2 macrophages, FK866 reduces oxidative phosphorylation, and NMN restores it.

“FK866-treated M2 macrophages had reduced oxidative phosphorylation that was rescued by addition of NMN (Fig. 2c)”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Adding NMN or NR restores NAD in M2 macrophages but not in M1 macrophages.

“both significantly restored NAD levels in M2, but not M1, macrophages”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

M1 polarization raises Nampt and Nmnat1 mRNAs but leaves Nmnat2, Nmnat3, and Ido1 mRNAs largely unchanged.

“In M1 macrophages, we observed increased levels of Nampt or Nmnat1 mRNAs, which maintain nuclear NAD levels (Fig. 1a), but no significant changes in the levels of Nmnat2 or Nmnat3 mRNAs, which regulate NAM salvage in the cytoplasm or mitochondria, respectively, or Ido1 mRNA (Fig. 1a).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

FK866 reduces both mRNA and secreted protein for M1 cytokines IL-6 and TNF-α, but not for Irg1.

“pretreatment of macrophages with FK866 also suppressed mRNA levels and secretion of M1 inflammatory cytokines, such as IL-6 and tumour necrosis factor-α (TNF-α), but not the metabolic enzyme Irg1 (Fig. 2f,g).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Cd38 knockout and M1 or M2 polarization do not change PARP or sirtuin expression.

“PARP and sirtuin expression was unaffected by Cd38 KO or M1 or M2 polarization (Fig. 3b).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Only M1 macrophages hydrolyse extracellular εNAD, and they do so less than whole lysates.

“Extracellular εNAD was hydrolysed only by M1 macrophages (Extended Data Fig. 3b, c), although to a lesser extent than whole cell lysates (Fig. 3d).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

M1 gene expression in Nampt Fl/FlCreER BMDMs changes only slightly, likely because NAMPT deletion is incomplete.

“Nampt Fl/FlCreER BMDM M1 gene expression was only slightly affected (Extended Data Fig. 2f), probably due to incomplete deletion of NAMPT (Extended Data Fig. 2d).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Relative to WT M1 macrophages, Cd38 KO M1 macrophages have significantly more NAD and NADP and a higher NAD/NAM ratio.

“Cd38 KO M1 macrophages also had significantly higher levels of NAD and NADP and a higher NAD/NAM ratio than did WT M1 macrophages (Fig. 3e,f).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

FK866 lowers mRNAs for M2 markers Arg1, Mgl2, Ccl24, and Retnlb, while Ym1 and Cd36 mRNAs remain unchanged.

“This treatment decreased mRNA levels of multiple M2 markers (arginase 1 (Arg1), galactose N-acetyl-galactosamine-specific lectin 2 (Mgl2), C–C motif chemokine ligand 24 (Ccl24) and resistin-like beta (Retnlb, also known as Fizz1)) (Fig. 2d), although the M2 markers Ym1 and Cd36 were unaffected.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

WT M1 macrophages contain more NAM and ADPR than Cd38 KO M1 macrophages, in which these metabolites fall to baseline.

“WT M1 macrophages had more NAM and ADPR, two products resulting from CD38 activity on NAD (Fig. 3e), whereas these metabolites were suppressed to basal levels in M1 macrophages from Cd38 KOs (Fig. 3e).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Lysates from Cd38 KO M1 macrophages have NADase activity close to baseline, unlike WT M1 lysates.

“lysates from Cd38 KO M1 macrophages showed near basal NADase activity compared to that in WT M1 macrophages (Fig. 3c, d).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

H2O2 causes detectable DNA damage in M0 and M2 macrophages, but WT and Cd38 KO M1 macrophages sustain less damage.

“treatment of macrophages with H2O2 induced detectable DNA damage in M0 and M2 macrophages (Extended Data Fig. 3h); however, both WT and Cd38 KO M1 macrophages showed less DNA damage”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

In aged versus young eWAT, mRNAs rise for CD38, p16 and p21 senescence markers, and the SASP cytokines Il6, Il1b, Il1a, and Il10.

“mRNAs encoding CD38, senescence markers (Cdkn2a mRNA, encoding p16Ink4a, and Cdkn1a mRNA encoding p21Cip1) and the inflammatory cytokines that comprise the SASP (Il6, Il1b, ll1a and Il10 mRNAs) increased in aged eWAT, as compared with their levels in young eWAT (Fig. 4b and Extended Data Fig. 4b).”

May combine two claims.

NADase activity is lower in Cd38 KO and Cd38/Cd157 DKO M1 macrophages, but Cd157 KO M1 macrophages keep activity near WT levels.

“whereas Cd38 KO and Cd38/Cd157 double-KO (DKO) M1 macrophages have attenuated NADase activity, Cd157 KO M1 macrophages retain NADase activity, similarly to WT M1 macrophages (Fig. 3g,h).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Old eWAT has more CD38 and CD157 protein and signs of greater inflammasome activation, including the 20-kDa caspase-1 form.

“old eWAT tissue contained greater protein levels of CD38 and CD157 (Fig. 4c,d), and noticeably elevated levels of inflammasome activation, as indicated by higher levels of the 20-kDa processed form of caspase-1 (Fig. 4c).”

May combine two claims.

Tabula Muris scRNA-seq shows CD38 expression in adipose-resident macrophages but not in non-resident macrophages.

“CD38 expression was specific to adipose-tissue-resident macrophages (CD68+CD64+F4/80+CD11b+CD11c−), not the non-resident population (CD68+CD64+F4/80+CD11b+CD11c+) (Fig. 4h)”

• cites [36] Consortium, T. T. M. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 562, 367–372 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

CD38 expression rises progressively with age in resident macrophages but not in non-resident macrophages.

“resident macrophages from older animals, but not non-resident macrophages, showed progressively increased CD38 expression (Fig. 4e and Extended Data Fig. 4f).”

May combine two claims.

In old eWAT, F4/80+ macrophages and p21+ senescent cells are more numerous and lie close together than in young eWAT.

“Further IF analysis showed an increase in both F4/80+ macrophages and p21+ senescent cells in close proximity to each other in old, compared with young, eWAT (Fig. 5b).”

May combine two claims.

Liver NAD levels fall with ageing, and this decline depends on CD38.

“Ageing is associated with a decline in liver NAD levels, which we can confirm is CD38 dependent (Fig. 6a) as has recently been reported7.”

• cites [7] Camacho-Pereira, J. et al. CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell Metab. 23, 1127–1139 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Sen(IR) preadipocytes have more SA-Bgal activity and higher p16 and p21 mRNAs, with only modest Cd38 expression.

“sen(IR)-preadipocytes, compared with non-senescent preadipocytes, showed elevated SA-Bgal activity (Fig. 5i) and higher levels of mRNAs encoding p16Ink4a and p21Cip, but only modest expression of Cd38 (Fig. 5j).”

• AssumptionRests on an assumption: Doxorubicin-induced and ionizing radiation-induced senescence model ageing-associated cellular senescence in vivo.

May combine two claims.

Sen(IR) fibroblasts show higher Csf1 transcripts and other growth factors, and proteomics confirms increased protein in conditioned medium.

“sen(IR)-fibroblasts had increased expression of Csf1 transcripts (Extended Data Fig. 5e) and other growth factors40, which we confirmed were significantly increased at the protein level by proteomic analysis of the conditioned medium (Extended Data Fig. 5g).”

• cites [40] Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010). sourceNot checked yet

  Not checked in the free run.

• AssumptionRests on an assumption: Doxorubicin-induced and ionizing radiation-induced senescence model ageing-associated cellular senescence in vivo.

May combine two claims.

In BMDMs, TNF-α, IL-6, and IL-10 raise Cd38 expression, whereas many other cytokines, including type 1 interferons, do not.

“although many of these cytokines including type 1 interferons did not promote Cd38 expression, TNF-α, IL-6 and IL-10 significantly increased Cd38 expression (Extended Data Fig. 5f).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Conditioned medium from senescent cells raises Cd38 and, less so, Cd157 in macrophages, without affecting other NAD-consuming enzyme genes.

“Macrophages that were cultured for 24 h in conditioned media from senescent cells upregulated Cd38, and to a lesser extent Cd157, but not other genes encoding NAD-consuming enzymes (Fig. 5f).”

• AssumptionRests on an assumption: Doxorubicin-induced and ionizing radiation-induced senescence model ageing-associated cellular senescence in vivo.
• AssumptionRests on an assumption: Conditioned medium from senescent cells reproduces the SASP effects on macrophages in vivo.

Probably not a substantial claim: may not pass S3 (load-bearing).

Liver scRNA-seq from Tabula Muris identifies eight distinct cell populations.

“Annotation of single-cell RNA-seq data from the livers of young and old (1–30 months of age) mice revealed 8 distinct cell populations in the liver (Fig. 6d).”

• cites [55] The Tabula Muris Consortium. A single-cell transcriptomic atlas characterizes ageing tissues in the mouse. Nature 583, 590–595 (2020).Not checked yet

  Not checked in the free run.

May combine two claims.

Immunofluorescence shows more CD38 in liver sinusoids and more F4/80+ macrophages near sinusoids in old versus young mice.

“IF staining for F4/80+ Kupffer cells and CD38 revealed increased expression of CD38 in liver sinusoids and greater numbers of F4/80+ macrophages near the sinusoids in old than in young mice (Fig. 6b).”

May combine two claims.

Aged Kupffer cells with higher Cd38 also show higher Nampt, Il1b, Il18, and Nfkbiz mRNAs, pointing to pro-inflammatory M1-like polarization.

“increased expression of Cd38 in aged Kupffer cells was paralleled by increased mRNA expression of the nicotinamide-salvage-pathway enzyme (Nampt mRNA), inflammatory cytokines (Il1b and Il18 mRNAs) and an inflammatory transcription factor (Nfkbiz mRNA), which is activated downstream of nuclear factor-κB, suggesting that Kupffer cells from old mice undergo a pro-inflammatory M1-like polarization.”

May combine two claims.

Old endothelial cells show only a modest Cd38 increase, and hepatocytes never express Cd38.

“expression of Cd38 in endothelial cells from old animals increased only modestly, and hepatocytes did not express Cd38 at any age (Extended Data Fig. 6a,b).”

May combine two claims.

Old mice have higher Cd38 expression in Kupffer cells, and more Kupffer cells express Cd38 as age increases.

“Cd38 gene expression was higher in Kupffer cells from old mice (24–30 months old), and the number of Kupffer cells that expressed Cd38 also increased with age (Fig. 6g-i)”

May combine two claims.

Acute LPS treatment elevates inflammatory cytokine expression, macrophage markers F4/80 but not Cd68, and Cd38 and Cd157 expression in eWAT.

“the eWAT of mice treated with an acute LPS dose also had significantly elevated expression of inflammatory cytokines (Fig. 7i and Extended Data Fig. 7e) and macrophage markers (F4/80 but not Cd68), and significant expression of Cd38 and Cd157 (Fig. 7i).”

May combine two claims.

In eWAT of LPS-injected mice, mRNA levels of inflammatory cytokines Tnfa and Il1b, macrophage markers Cd68 and F4/80, and NAD hydrolases Cd38 and Cd157 are higher than in PBS-injected mice.

“Further analysis of eWAT from mice injected with LPS showed higher levels of inflammatory cytokine (Tnfa and Il1b) (Fig. 7e), macrophage marker (Cd68 and F4/80) and NAD hydrolase (Cd38 and Cd157) mRNAs (Fig. 7e), which also increased at the protein level (Extended Data Fig. 7c), than in eWAT of mice injected with PBS.”

May combine two claims.

Chronic LPS treatment does not increase, and even decreases, CD38 expression in other immune-cell populations, including B and T cells.

“whereas CD38 expression was not increased, and was even decreased, in other immune-cell populations (see Extended Data Fig. 7a for gating strategies), including B and T cells (Extended Data Fig. 7b).”

May combine two claims.

CD38 and CD157 protein levels are also increased in eWAT of LPS-injected mice.

“which also increased at the protein level (Extended Data Fig. 7c)”

May combine two claims.

NA increases and nicotinic acid dinucleotide declines in both eWAT and liver of WT and Cd38 KO mice treated with LPS.

“NA increased and nicotinic acid dinucleotide declined in both the eWAT and liver of WT and Cd38 KO mice treated with LPS (Extended Data Fig. 7g,h)”

May combine two claims.

The doxorubicin-induced rise in senescence markers is accompanied by higher CD38 expression, which is reversed when senescent cells are deleted with ganciclovir.

“Notably, this increase was paralleled by increased CD38 expression, which was reversed after deleting senescent cells with GCV (Fig. 6k).”

May combine two claims.

Doxorubicin treatment of p16-3MR mice increases expression of the senescence markers p16Ink4a and p21Cip1 and the mRFP reporter.

“We treated p16-3MR mice with doxo, which, as expected, increased expression of the senescent-cell markers p16Ink4a and p21Cip1 along with mRFP reporter (Fig. 6k).”

May combine two claims.

In livers of old mice, SA-β-gal activity is higher than in young mice, indicating more senescent cells.

“SA-Bgal activity increased in the livers of old versus young mice, indicative of an increase in senescent cells (Fig. 6j).”

May combine two claims.

In eWAT of LPS-treated mice, Parp1 and Parp2 expression and PARP activity are not enhanced, whereas basal PARylation is increased in Cd38 KO mice.

“we did not detect enhanced expression of Parp1 or Parp2, or any detectable PARP activity, in the eWAT of LPS-treated mice, whereas basal PARylation was noticeably increased in Cd38 KO mice (Fig. 7i and Extended Data Fig. 7e,f).”

May combine two claims.

Cd38 KO mice treated with LPS have significantly less liver NAD than PBS-treated Cd38 KO mice, but still significantly more NAD than LPS-treated WT mice.

“Cd38 KO mice treated with LPS had significantly less NAD than PBS-treated Cd38 KO mice, although they still had signficantly higher NAD levels than LPS-treated WT mice (Fig. 7l).”

May combine two claims.

Only WT LPS-treated mice show increased MeNAM production in both eWAT and liver; WT PBS-treated and Cd38 KO mice do not.

“only WT LPS-treated mice showed increased production of MeNAM in both eWAT and liver; WT PBS-treated mice and Cd38 KO mice did not (Fig. 7j,l).”

May combine two claims.

LPS exposure increases Nnmt expression in liver, but not eWAT, in both WT and Cd38 KO mice.

“LPS exposure increased the expression of Nnmt in the liver, but not eWAT, in both WT and Cd38 KO mice (Fig. 7k and Extended Data Fig. 7e).”

May combine two claims.

In eWAT, oxNAM abundance is tenfold less than MeNAM abundance, and oxNAM is undetectable in liver.

“the overall abundance of oxNAM was tenfold less than that of MeNAM in the eWAT (Fig. 7j), and we were unable to detect oxNAM in the liver.”

May combine two claims.

Cd38 KO tissue has enhanced basal PARylation in BMDMs and eWAT, similar to enhanced SIRT3 activity reported in Cd38 KO mice.

“one of the most prominent phenotypes in Cd38 KO tissue is the enhanced basal PARylation in Cd38 KO BMDMs (Extended Data Fig. 3g) and eWAT (Extended Data Fig. 7f), similar to the enhanced SIRT3 activity that was reported in Cd38 KO mice7.”

May combine two claims.

2py and 4py metabolites increase in livers of WT LPS-treated mice, but not in Cd38 KO mice.

“Both 2py and 4py metabolites increased in the livers of WT LPS-treated mice, but not in Cd38 KO mice.”

Classified as probably not a substantial claim.

CD38 expression increases in visceral white adipose tissue during ageing.

“a recent report has shown an increased expression of CD38 during ageing in visceral white adipose tissue7.”

• cites [7] Camacho-Pereira, J. et al. CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell Metab. 23, 1127–1139 (2016). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Most cells are thought to maintain intracellular NAD mainly by recycling NAM through NAMPT.

“the prevailing thought is that the recycling of NAM via nicotinamide phosphoribosyltransferase (NAMPT) is the predominant pathway used by most cells to maintain intracellular NAD levels6.”

• cites [6] Liu, L. et al. Quantitative analysis of NAD synthesis-breakdown fluxes. Cell Metab. 27, 1067–1080 e1065 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

NAD precursor supplementation with NR, NAM, NA, and NMN mitigates several age-associated diseases.

“restoration of NAD levels with the NAD precursor vitamins nicotinamide riboside (NR), nicotinamide (NAM) and nicotinic acid (NA), in addition to the biosynthetic NAD precursor nicotinamide mononucleotide (NMN), appears to mitigate several age-associated diseases2-4.”

• cites [2] Yoshino, J., Baur, J. A. & Imai, S. I. NAD+ intermediates: the biology and therapeutic potential of NMN and NR. Cell Metab. 27, 513–528 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [3] Mitchell, S. J. et al. Nicotinamide improves aspects of healthspan, but not lifespan, in mice. Cell Metab. 27, 667–676 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [4] Verdin, E. NAD+ in aging, metabolism, and neurodegeneration. Science 350, 1208–1213 (2015). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

Immune cells express CD38, and inflammatory states raise its expression.

“CD38 is ubiquitously expressed by immune cells, and its expression increases during inflammatory conditions8-10.”

• cites [8] Jackson, D. G. & Bell, J. I. Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation. J. Immunol. 144, 2811–2815 (1990). sourceNot checked yet

  Not checked in the free run.

• cites [9] Schneider, M. et al. CD38 is expressed on inflammatory cells of the intestine and promotes intestinal inflammation. PLoS ONE 10, e0126007 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [10] Savarino, A., Bottarel, F., Malavasi, F. & Dianzani, U. Role of CD38 in HIV-1 infection: an epiphenomenon of T-cell activation or an active player in virus/host interactions? AIDS 14, 1079–1089 (2000). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Mice without CD38 resist age-related NAD loss and show better metabolic health and SIRT3-dependent mitochondrial function.

“mice lacking CD38 (Cd38 KO) were protected from age-related NAD decline and had enhanced metabolic health and sirtuin 3 (SIRT3)-dependent mitochondrial function”

• cites [7] Camacho-Pereira, J. et al. CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell Metab. 23, 1127–1139 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

No tryptophan-pathway metabolites beyond kynurenine were detected in any macrophage polarization state, suggesting tryptophan is not a de novo NAD source in these cells.

“We were unable to detect tryptophan-pathway metabolites downstream of kynurenine in M0, M1 or M2 macrophages, which suggests that tryptophan may not contribute to de novo NAD synthesis in macrophages, regardless of polarization state.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

Macrophage metabolic reprogramming requires NAD, and NAD fine-tunes a subset of M1 and M2 genes.

“we provide evidence that NAD is necessary to support macrophage metabolic reprogramming and fine-tunes the expression of a subset of M1 and M2 genes.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

CD38 acts on NAD both outside and inside cells, with much of its NADase activity in macrophages occurring intracellularly.

“These data demonstrate that CD38 cleaves NAD outside and inside cells, and that a substantial fraction of CD38 NADase activity in macrophages is intracellular.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Even low CD38 expression may affect NAD in quiescent and M2 macrophages, possibly through another NAD-consuming enzyme compensating.

“which suggests that basal expression of CD38 may impact NAD levels in quiescent and M2 macrophage subsets, perhaps due to compensation from another NAD-consuming enzyme.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

NAD levels in macrophages are controlled by NAM salvage, and NAD is a key regulator of polarization and of a subset of M1 and M2 gene mRNAs.

“These results show that the NAM-salvage pathway regulates macrophage NAD levels, and that NAD is a crucial regulator of macrophage polarization and mRNA abundance of a subset of M2 and M1 genes.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

May combine two claims.

M1 and M2 macrophages differ in how much NAD they need; M2 cells need more for M2 gene expression and effector functions.

“they show that M1 and M2 macrophages have different requirements for NAD levels, with M2 macrophages requiring higher levels to support M2 gene expression and immune effector functions (Fig. 2h).”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

Probably not a substantial claim: may not pass S3 (load-bearing).

M1 macrophages may raise antioxidant genes to survive and avoid ROS-induced DNA damage.

“These data are consistent with the hypothesis suggesting that M1 macrophages upregulate antioxidant genes as a survival mechanism and are protected from the DNA-damaging effects of ROS31.”

• cites [31] Virag, L., Jaen, R. I., Regdon, Z., Bosca, L. & Prieto, P. Self-defense of macrophages against oxidative injury: fighting for their own survival. Redox Biol. 26, 101261 (2019). sourceNot checked yet

  Not checked in the free run.

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.

Probably not a substantial claim: may not pass S3 (load-bearing).

A subset of tissue-resident macrophages co-expresses Lyve1, Fcna, and Cd38.

“Recent reports have characterized gene expression in tissue-resident immune cells and found that Lyve1 and Fcna are expressed along with Cd38 by a subset of tissue-resident macrophages37,38”

• cites [37] Chakarov, S. et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science 363, eaau0964 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [38] Mrdjen, D. et al. High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease. Immunity 48, 380–395 e386 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Old adipose tissue is highly inflamed, contains more senescent cells and macrophages, and shifts resident macrophages from M2 toward a pro-inflammatory M1-like state with higher CD38.

“adipose tissue from old animals was highly inflamed, had more senescent cells and macrophages and showed a noticeable shift in tissue-resident macrophage polarization from a M2 to a pro-inflammatory M1-like state with increased CD38 expression.”

May combine two claims.

In ageing eWAT, senescent cells and the SASP may drive higher CD38 expression in adipose macrophages.

“These results suggest that senescent cells and the SASP might be responsible for increasing CD38 expression in adipose-tissue macrophages in ageing eWAT.”

• AssumptionRests on an assumption: Doxorubicin-induced and ionizing radiation-induced senescence model ageing-associated cellular senescence in vivo.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Kupffer cells make up as much as 15% of liver cells and 80-90% of tissue-resident macrophages.

“Liver-resident macrophages, known as Kupffer cells, comprise up to 15% of total liver cells and 80–90% of all tissue-resident macrophages53.”

• cites [53] Jager, J., Aparicio-Vergara, M. & Aouadi, M. Liver innate immune cells and insulin resistance: the multiple facets of Kupffer cells. J. Intern. Med. 280, 209–220 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Such M1-like macrophages have high CD38 and stronger CD38-dependent NADase activity, which lowers tissue NAD.

“These M1-like macrophages express high levels of the NAD-consuming enzyme CD38 and have enhanced CD38-dependent NADase activity, thereby reducing tissue NAD levels.”

• AssumptionRests on an assumption: In vitro macrophage polarization states (M0, M1, M2) correspond to macrophage states in vivo.
• AssumptionRests on an assumption: Bone-marrow-derived macrophages model tissue-resident macrophages such as adipose macrophages and Kupffer cells.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Pro-inflammatory Kupffer cell activation is thought to contribute to age-related liver disease and to infectious diseases such as hepatitis.

“Activation of Kupffer cells to a pro-inflammatory state is thought to be a driver not only of ageing-related liver disease, such as non-alcoholic liver disease, cirrhosis and fibrosis, but also in the pathogenesis of infectious diseases such as hepatitis54.”

• cites [54] Stahl, E. C., Haschak, M. J., Popovic, B. & Brown, B. N. Macrophages in the aging liver and age-related liver disease. Front. Immunol. 9, 2795 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

SASP cytokines from senescent cells drive macrophages to multiply and to express CD38.

“inflammatory cytokines secreted by senescent cells (the senescence-associated secretory phenotype, SASP) induce macrophages to proliferate and express CD38.”

• AssumptionRests on an assumption: Doxorubicin-induced and ionizing radiation-induced senescence model ageing-associated cellular senescence in vivo.
• AssumptionRests on an assumption: Conditioned medium from senescent cells reproduces the SASP effects on macrophages in vivo.

May combine two claims.

In livers of doxorubicin-treated mice, senescent-cell buildup is both enough and required for the rise in CD38 expression.

“Thus, the accumulation of senescent cells in the liver of doxo-treated mice is both sufficient and necessary to drive enhanced CD38 expression.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CD38 regulates immune-cell homing, innate immune responses to pathogens and antitumour immunity.

“Previous studies have reported that CD38 regulates immune-cell homing, innate immune responses to pathogens and antitumour immunity63-65.”

• cites [63] Chatterjee, S. et al. CD38–NAD+ axis regulates immunotherapeutic anti-tumor T cell response.Cell Metab. 27, 85–100 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [64] Lischke, T. et al. CD38 controls the innate immune response against Listeria monocytogenes. Infect. Immun. 81, 4091–4099 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [65] Partida-Sanchez, S. et al. Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by ADP-ribose, the major product generated by the CD38 enzyme reaction. J. Immunol. 179, 7827–7839 (2007). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Intestinal-barrier permeability increases during ageing, leading to higher endotoxin levels, including LPS, in ageing tissues.

“A recent study has shown that intestinal-barrier permeability increases during ageing, leading to higher levels of endotoxins, including LPS, in ageing tissues58,59.”

• cites [58] Buford, T. W. (Dis)Trust your gut: the gut microbiome in age-related inflammation, health, and disease. Microbiome 5, 80 (2017). sourceNot checked yet

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• cites [59] Thevaranjan, N. et al. Age-associated microbial dysbiosis promotes intestinal permeability, systemic inflammation, and macrophage dysfunction. Cell Host Microbe 21, 455–466 (2017). sourceNot checked yet

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May combine two claims.

The SASP is both required and enough to raise CD38 expression in macrophages and to drive their proliferation, producing more CD38+ macrophages in aged mice and in young mice given doxorubicin to increase senescent-cell burden.

“Here, we provide evidence that the SASP is necessary and sufficient to increase CD38 expression in macrophages and promote macrophage proliferation, leading to an increase in CD38+ macrophages in aged mice and young mice with an increased senescent-cell burden due to treatment with the genotoxic agent doxo (Figs. 5 and 6).”

• AssumptionRests on an assumption: As organisms age, genotoxic and other stressors cause some cells to undergo senescence.

Probably not a substantial claim: may not pass S3 (load-bearing).

In aged mice, senescent cells have been implicated in liver inflammation characterized by fat deposition and greater macrophage numbers.

“In aged mice, senescent cells have recently been implicated in liver inflammation, characterized by fat deposition and greater numbers of macrophages56.”

• cites [56] Ogrodnik, M. et al. Cellular senescence drives age-dependent hepatic steatosis. Nat. Commun. 8, 15691 (2017). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The methylation and removal of surplus NAM made by CD38 in the liver might also affect NAD levels in peripheral tissues during inflammation.

“Thus, the methylation and clearance of excess NAM, derived from CD38 in the liver, may also influence NAD levels in peripheral tissues under inflammatory conditions.”

• AssumptionRests on an assumption: NNMT converts NAM to MeNAM, diverting NAM from the NAM-salvage pathway and affecting global NAD+ levels.
• AssumptionRests on an assumption: MeNAM can be oxidized by AOX to 2py and 4py, which are secreted in urine.

May combine two claims.

Greater CD38-driven NAD turnover or lower NAD biosynthesis alone cannot fully account for the NAD drop after LPS, since the NAM-salvage pathway ought to turn surplus NAM into NAD.

“Enhanced NAD turnover by CD38 or decreased NAD biosynthesis does not fully explain why NAD levels decline in response to LPS, because the NAM-salvage pathway should be able to convert excess NAM to NAD (Fig. 7m).”

• AssumptionRests on an assumption: NNMT converts NAM to MeNAM, diverting NAM from the NAM-salvage pathway and affecting global NAD+ levels.

Probably not a substantial claim: may not pass S3 (load-bearing).

CD38 may act as a feedback mechanism to initiate resolution of acute inflammation.

“suggesting that CD38 may act as a feedback mechanism to initiate the resolution of acute inflammation.”

• AssumptionRests on an assumption: NNMT converts NAM to MeNAM, diverting NAM from the NAM-salvage pathway and affecting global NAD+ levels.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Senescent cells secrete inflammatory and other factors that can disrupt normal cellular functions, damage tissues and promote carcinogenesis.

“they also secrete inflammatory and other factors that can disrupt normal cellular functions, damage tissues and promote carcinogenesis61,62.”

• cites [61] Campisi, J. & Robert, L. Cell senescence: role in aging and age-related diseases. Interdiscip. Top. Gerontol. 39, 45–61 (2014). sourceNot checked yet

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• cites [62] Campisi, J. Aging, cellular senescence, and cancer. Annu Rev. Physiol. 75, 685–705 (2013). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Outside the liver, NAM is the major NAD precursor used by peripheral cells and tissues to maintain NAD levels.

“a recent study using stable-isotope tracing has demonstrated that, outside the liver, NAM is the major NAD precursor used by peripheral cells and tissues to maintain NAD levels6.”

• cites [6] Liu, L. et al. Quantitative analysis of NAD synthesis-breakdown fluxes. Cell Metab. 27, 1067–1080 e1065 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CD38 may be expressed by inflammatory M1 macrophages to modulate cell-autonomous NAD-dependent processes controlling macrophage activation and gene expression, or metabolic pathways in neighbouring cells.

“Thus, CD38 may be expressed by inflammatory M1 macrophages to modulate cell-autonomous NAD-dependent processes to control macrophage activation and gene expression (Fig. 2), or metabolic pathways in neighbouring cells.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

All procedures were approved by the Buck Institute and UCSF Gladstone IACUC.

“All procedures were performed in accordance with federal guidelines and those set forth by the Buck Institute and the UCSF Gladstone Institutional Animal Care and Use Committees (IACUC).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Littermates were not used to compare WT and Cd38 KO mice.

“Littermates were not used to compare WT and Cd38 KO mice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The cells used for senescence experiments were approximately 85% preadipocytes, as confirmed by flow cytometry.

“The trypsinized cells (~ 85% PAs) were confirmed by flow cytometry”

Probably not a substantial claim: may not pass S3 (load-bearing).

Excess NAM is oxidized by cytochrome P450 enzyme CYP2E to NAM-N-oxide (oxNAM), which is secreted in urine.

“excess NAM was reported to be oxidized by the cytochrome P450 enzyme CYP2E, converting NAM to NAM-N-oxide (oxNAM), which is also secreted in urine60 (Fig. 7m).”

• cites [60] Real, A. M., Hong, S. & Pissios, P. Nicotinamide N-oxidation by CYP2E1 in human liver microsomes. Drug Metab. Dispos. 41, 550–553 (2013). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Neural network training used data augmentation to prevent overfitting and improve robustness.

“All training sets were extended with data augmentation, where the set of images was automatically and randomly manipulated during training (by adjusting size, brightness and rotation) to prevent overfitting and improve robustness.”

Probably not a substantial claim: may not pass S3 (load-bearing).

CD38 influences cellular processes and immune responses by producing secondary messenger NAD metabolites, including cADPR and NaADP, to control Ca2+ mobilization and cell migration.

“Another mechanism by which CD38 may influence cellular processes and immune responses is by the production of secondary messenger NAD metabolites, including cADPR and NaADP, to control Ca2+ mobilization and cell migration67.”

• cites [67] Wei, W., Graeff, R. & Yue, J. Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway. World J. Biol. Chem. 5, 58–67 (2014). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

NAM has immunomodulatory properties, and high NAM levels can reduce inflammatory cytokine production in macrophages.

“NAM has immunomodulatory properties, and high NAM levels can reduce inflammatory cytokine production in macrophages68”

• cites [68] Weiss, R. et al. Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features. Innate Immun. 21, 813–826 (2015). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Chini and colleagues reported similar findings consistent with inflammaging as a driver of NAD decline during ageing.

“Chini and colleagues have reported similar findings, which are consistent with the role of inflammaging as a driver of NAD decline during ageing69.”

• cites [69] Chini, C. C. S. et al. CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels. Nat. Metab. 2, 1284–1304 (2020). sourceNot checked yet

  Not checked in the free run.