\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{NAD\textsuperscript{+} modulation of intestinal macrophages renders
anti-inflammatory functionality and ameliorates gut inflammation}



\author[1]{Young-In Kim}%
\author[2]{Inseok Ko}%
\author[1]{Eun-Je Yi}%
\author[2]{Jusik Kim}%
\author[2]{Yong Rae Hong}%
\author[2]{Wheeseong Lee}%
\author[1]{Sun-Young  Chang}%
\affil[1]{Ajou University}%
\affil[2]{Lmito Therapeutics}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose: Macrophages not only can maintain gut immune
homeostasis by driving clearance of infection, but also can prevent
chronic inflammation and induce tissue repair. Macrophages are a
heterogenous group of cells whose characteristics are determined by
tissue microenvironment and metabolism. Since macrophages play an
important role in inflammatory disorders such as inflammatory bowel
disease (IBD), they can be a potential therapeutic target. Experimental
Approach: Here we show an IBD therapeutic candidate LMT503, a substrate
which modulates NADH quinone oxidoreductase 1, which enhances
NAD\textsuperscript{+} and induce anti-inflammatory macrophage
polarization. To determine the anti-inflammatory effect of LMT503, a
dextran sulfate sodium (DSS)-induced colitis mouse model was used in
this study. Key Results: Treatment of bone marrow derived macrophages
(BMDMs) with LMT503 increased IL-10 and Arg1 levels but decreased levels
of TNF-\selectlanguage{greek}α, \selectlanguage{english}iNOS, and IL-6. LMT503 also increased levels of SIRT1, SIRT3,
and SIRT6, suggesting that macrophages were driven to an
anti-inflammatory character. In a murine DSS-induced colitis model, oral
treatment with LMT503 ameliorated colonic inflammation and decreased
infiltrating monocytes and neutrophils. Although NAD\textsuperscript{+}
enhancement did not alter
CX\textsubscript{3}CR1\textsuperscript{int}CD206\textsuperscript{-} or
CX\textsubscript{3}CR1\textsuperscript{hi}CD206\textsuperscript{+} colon
macrophage population, it decreased levels of TNF-\selectlanguage{greek}α \selectlanguage{english}and iNOS and
increased IL-10 level, with colonic macrophages showing an
anti-inflammatory character shift. Depletion of CX\textsubscript{3}CR1
expressing gut resident macrophages abrogated the immune regulatory
effect of LMT503 in the colon. Conclusion and Implications: These data
suggest that LMT503 is a therapeutic candidate that can target
macrophages to drive polarization with an immunosuppressive character
and ameliorate IBD.%
\end{abstract}%



\sloppy


\textbf{NAD\textsuperscript{+} modulation of intestinal macrophages
renders anti-inflammatory functionality and ameliorates gut
inflammation}

Young-In Kim\textsuperscript{1,2,*}, Inseok Ko\textsuperscript{3,*},
Eun-Je Yi\textsuperscript{1}, Jusik Kim\textsuperscript{3}, Yong Rae
Hong\textsuperscript{3, \#}, Wheeseong Lee\textsuperscript{3}, Sun-Young
Chang\textsuperscript{1}

\textsuperscript{1}Laboratory of Microbiology, College of Pharmacy, and
Research Institute of Pharmaceutical Science and Technology (RIPST),
Ajou University, Suwon, Republic of Korea

\textsuperscript{2}Korea Initiative for fostering University of Research
and Innovation (KIURI) Program, Ajou University School of Medicine,
Suwon, Republic of Korea

\textsuperscript{3}Lmito Therapeutics, Yongin-si, Republic of Korea

\textsuperscript{*}These authors contributed equally to this work

\textsuperscript{\#}Current address: Autotelic Bio, Yongin-si, Republic
of Korea

Corresponding author: Dr. Sun-Young Chang, Laboratory of Microbiology,
College of Pharmacy, Ajou University, Worldcupro 206, Yeongtong-gu,
Suwon, Gyeonggi-do 16499, Republic of Korea., Tel: +82-31-219-3454, Fax:
+82-31-219-3435, E-mail:\emph{sychang@ajou.ac.kr}

\textbf{Short title:} NAD\textsuperscript{+} enhancement of macrophages
ameliorates gut inflammation

\textbf{Abstract}

Background and Purpose:

Macrophages not only can maintain gut immune homeostasis by driving
clearance of infection, but also can prevent chronic inflammation and
induce tissue repair. Macrophages are a heterogenous group of cells
whose characteristics are determined by tissue microenvironment and
metabolism. Since macrophages play an important role in inflammatory
disorders such as inflammatory bowel disease (IBD), they can be a
potential therapeutic target.

Experimental Approach:

Here we show an IBD therapeutic candidate LMT503, a substrate which
modulates NADH quinone oxidoreductase 1, which enhances
NAD\textsuperscript{+} and induce anti-inflammatory macrophage
polarization. To determine the anti-inflammatory effect of LMT503, a
dextran sulfate sodium (DSS)-induced colitis mouse model was used in
this study.

Key Results:

Treatment of bone marrow derived macrophages (BMDMs) with LMT503
increased IL-10 and Arg1 levels but decreased levels of TNF-\selectlanguage{greek}α, \selectlanguage{english}iNOS, and
IL-6. LMT503 also increased levels of SIRT1, SIRT3, and SIRT6,
suggesting that macrophages were driven to an anti-inflammatory
character. In a murine DSS-induced colitis model, oral treatment with
LMT503 ameliorated colonic inflammation and decreased infiltrating
monocytes and neutrophils. Although NAD\textsuperscript{+} enhancement
did not alter
CX\textsubscript{3}CR1\textsuperscript{int}CD206\textsuperscript{-} or
CX\textsubscript{3}CR1\textsuperscript{hi}CD206\textsuperscript{+} colon
macrophage population, it decreased levels of TNF-\selectlanguage{greek}α \selectlanguage{english}and iNOS and
increased IL-10 level, with colonic macrophages showing an
anti-inflammatory character shift. Depletion of CX\textsubscript{3}CR1
expressing gut resident macrophages abrogated the immune regulatory
effect of LMT503 in the colon.

Conclusion and Implications:

These data suggest that LMT503 is a therapeutic candidate that can
target macrophages to drive polarization with an immunosuppressive
character and ameliorate IBD.

\textbf{Keywords:} Inflammatory bowel disease,
NAD\textsuperscript{+}modulation, Gut macrophage, Macrophage
polarization, Colon

\textbf{Abbreviations:}

Arg1: arginase 1

BMDM: bone marrow derived macrophage

DSS: dextran sulfate sodium

IBD: Inflammatory bowel disease

IL: interleukin

iNOS: inducible nitric oxide synthase

MLN: mesenteric lymph node

NQO1: NADH quinone oxidoreductase 1

PBMC: peripheral blood mononuclear cell

PP: Peyer's Patches

SIRT: Sirtuin; silent information regulator

TNF-\selectlanguage{greek}α: \selectlanguage{english}tumor necrosis factor \selectlanguage{greek}α



\selectlanguage{english}\textbf{What is already known}

Distinct metabolic pathways determine macrophage subtype and function.

\textbf{What does this study add}

Enhancement of NAD\textsuperscript{+} in gut macrophages shifted to an
anti-inflammatory character and ameliorated inflammation in a murine
colitis model.

\textbf{What is the clinical significance}

Modulation of NAD\textsuperscript{+} in macrophages may be a promising
therapeutic target to treat inflammatory bowel disease.



\textbf{Introduction}

The intestine not only serves as a barrier against external harmful
agents and pathogen infection, but also interacts with commensal
microbiome and food antigens. Therefore, the intestinal immune system
reacts to both inner and external environments to maintain homeostasis.
When this balance is disrupted, chronic inflammatory disorders can occur
in the intestine, leading to inflammatory bowel disease (IBD) (Maloy \&
Powrie, 2011). There are two main forms of IBD: Crohn's disease, which
includes inflammatory disorder throughout the gastrointestinal tract,
and ulcerative colitis, which is restricted in the colon (Kaser,
Zeissig, \& Blumberg, 2010). Causes of IBD is complicated. It involves
multiple factors such as genetic factors, failure of the host immune
system, and disruption of gut microbiota (Hill \& Artis, 2010; Podolsky,
1991).

Macrophages are innate immune cells well distributed in almost all
tissues and are highly heterogenous to achieve niche-specific functions.
Gut macrophages not only can maintain immune homeostasis by driving
clearance of infection, but also can control oral tolerance and tissue
repair (Mazzini, Massimiliano, Penna, \& Rescigno, 2014; Murray \& Wynn,
2011). Gut macrophages initiate inflammation in response to stimuli such
as pathogen or cell damage. After an inflammatory response takes place,
complete resolution must take place to maintain homeostasis. Gut
macrophages are key players initiating inflammation to stimuli such as
infection, but also in resolution of inflammation afterwards.
Dysfunction of macrophages may result in failure of resolution, leading
to chronic inflammation (Na, Stakenborg, Seok, \& Matteoli, 2019).
Alternatively activated macrophages can produce anti-inflammatory
cytokines IL-10 and growth factors such as TGF-\selectlanguage{greek}β, \selectlanguage{english}prostaglandin
E\textsubscript{2} (PGE\textsubscript{2}), bone morphogenetic protein 2
(BMP2), and WNT ligands (Lin et al., 2010; Wynn \& Vannella, 2016).
IL-10 production is known to promote immunosuppressive
CD4\textsuperscript{+} regulatory T cells (Treg).

Gut macrophages contain self-maintaining resident macrophages derived
from embryonic birth and bone marrow derived macrophages matured from
circulating monocytes (Viola \& Boeckxstaens, 2021). Freshly supplied
bone marrow derived Ly6C\textsuperscript{hi} monocytes undergo
differentiation into
CX\textsubscript{3}CR1\textsuperscript{+}macrophages in the lamina
propria (Bain et al., 2014). They rapidly upregulate major
histocompatibility complex type II (MHCII) and downregulate Ly6C. During
colitis, CX\textsubscript{3}CR1\textsuperscript{int} expressing
monocytes are recruited to the gut. They show a pro-inflammatory
phenotype. Mature CX\textsubscript{3}CR1\textsuperscript{hi} macrophages
are rather anti-inflammatory. They can maintain homeostasis (Diehl et
al., 2013; Geissmann, Jung, \& Littman, 2003; Viola \& Boeckxstaens,
2021).

IBD patients show altered differentiation of macrophages compared to
healthy individuals. Since functional macrophage defect can lead to
chronic inflammatory disorders, macrophages can be considered a
potential target for IBD treatment. Previous studies have shown that
macrophages have distinct phenotypes through metabolic regulation.
Inflammatory macrophages show enhanced glycolysis while
anti-inflammatory macrophages show enhanced oxidative phosphorylation
(Russell, Huang, \& VanderVen, 2019; Wculek et al., 2023). Distinct
pathways of arginine metabolism determine macrophage characters.
Inflammatory macrophages express nitric oxide synthase which leads to
production of nitric oxide and citrulline, while anti-inflammatory
macrophages express arginase which leads to production of urea and
ornithine (Rath, Muller, Kropf, Closs, \& Munder, 2014). Also,
inflammatory macrophages can be induced by accumulation of citrate or
succinate due to impairment of the TCA cycle (Harber et al., 2020;
Palmieri et al., 2020). Thus, utilizing metabolic modulation to induce
macrophage polarization can be a therapeutic approach to treat IBD.

In this study, we introduce LMT503, an IBD therapeutic candidate, which
act as a substrate for NADH quinone oxidoreductase 1 (NQO1), a
multi-functional protein that can increase cellular
NAD\textsuperscript{+}, drive oxidation-reduction reaction, and
stabilize proteins such as p53 (Nebert, Roe, Vandale, Bingham, \&
Oakley, 2002; Ross \& Siegel, 2021). Increased NAD\textsuperscript{+} by
NQO1 can activate sirtuins and drive metabolic modulation of macrophages
to have an anti-inflammatory character (S. Y. Park et al., 2017). The
objective of this study was to determine whether LMT503 could induce
polarization of macrophages to an immune-suppressive type to alleviate
colitis in a DSS-induced murine model.

\textbf{Methods}

\textbf{Cell culture and stimulation}

To prepare bone marrow derived macrophages (BMDMs), marrow was flushed
out from femurs and tibiae of hind legs using complete RPMI medium
(Invitrogen, Carlsbad, CA, USA). After red blood cells were lysed, cells
were seeded onto cell culture plates in complete RPMI medium with 20
ng/ml M-CSF (R\&D Systems, Minneapolis, MN, USA) and cultured for 6
days. To prepare monocytes from human peripheral blood mononuclear
cells, whole blood was layered on top of Histopaque 1077 (Sigma-Aldrich,
St. Louis, MO, USA) and centrifuged at 400 x g for 30 mins at room
temperature. The interface containing mononuclear cells was collected.
Monocytes were then selected using a MagniSort Human Monocyte Enrichment
Kit (Invitrogen, Carlsbad, CA, USA). Cells were stimulated with 200
ng/ml LPS for 4 hours and then treated with 10 or 100 \selectlanguage{greek}μ\selectlanguage{english}M LMT503 for 18
hours at 37\selectlanguage{ngerman}°C. For IL-1\selectlanguage{greek}β, \selectlanguage{english}BMDMs were stimulated with 200 ng/ml LPS for 3
hours followed by 5 mM ATP (Sigma-Aldrich, St. Louis, MO, USA) for 1
hour, and then treated with 10 or 100 \selectlanguage{greek}μ\selectlanguage{english}M LMT503 for 18 hours at 37\selectlanguage{ngerman}°C.
For monocytes derived from human peripheral blood mononuclear cells,
fresh blood was collected from healthy adult volunteers from Ajou
University Hospital. This study was approved by the Human Research
Protection Center of Ajou University Hospital (IRB \#2022-0534-001).

\textbf{NQO1 activity assay}

BMDMs were cultured with 200 ng/ml LPS and 10 or 100 \selectlanguage{greek}µ\selectlanguage{english}M LMT503 for 18
hours at 37\selectlanguage{ngerman}°C. Cells were washed with ice cold PBS. Cell pellet was then
used for analysis with a NQO1 Activity Assay Kit (Abcam, Cambridge, UK)
according to the manufacturer's instructions.

\textbf{Animals}

Dextran sulfate sodium (DSS)-induced colitis experiment was approved by
the Institutional Animal Care and Use Committee of Ajou University
(IACUC \#2023-0040). Animals were kept in the Laboratory Animal Research
Center of Ajou University Medical Center under specific pathogen-free
conditions. They received sterilized food and water \emph{ad libitum} .
Six-week-old wild-type (WT) C57BL/6 female mice were purchased from
Orient Bio Inc. (Sungnam, Korea). CX\textsubscript{3}CR1-DTR mice
(Jackson Laboratories) were kindly provided by D.R. Littman (New York
University Medical Center) (Diehl et al., 2013).

\textbf{Colitis}

Seven-week-old C57BL/6 female mice with initial body weight of 19 ± 1 g
were used in this study. To prepare DSS-induced colitis mouse model, 2\%
DSS (molecular weight 36,000--50,000 Da; MP Biomedicals LLC, Solon, OH,
USA) dissolved in autoclaved drinking water was provided to mice\emph{ad
libitum} for 5 days. Mice were then provided with normal drinking water
until the end of the experiment. Each dose of LMT503 (Korean patent
10-2022-0125359, Lmito Therapeutics, Sungnam, Republic of Korea) or
tofacitinib (Selleck Chemicals, Houston, TX) in 0.5\% methyl
cellulose/0.2\% Tween80 (Sigma-Aldrich, St. Louis, MO, USA) was used to
treat mice by oral gavage once every day starting from 5 days after DSS
treatment. During the whole period, body weight and disease activity
index (body weight loss, rectal bleeding, and diarrhea) were monitored
daily and scored as described previously (Kim et al., 2020). At day 10,
colon lengths were measured, and colon histology studies were proceeded.
To determined bacterial translocation, the spleen and mesenteric lymph
node (MLN) isolated from mice at day 10 were homogenized and serially
diluted. Serial dilutions were plated onto LB agar plates and incubated
at 37°C overnight. CFU/g was then determined. For mice
CX\textsubscript{3}CR1\textsuperscript{+} macrophage depletion,
CX\textsubscript{3}CR1-DTR mice received \emph{i.p.} injection of 200 ng
diphtheria toxin at day 1, day 4, and day 7 of DSS treatment.

\emph{Salmonella} Typhimurium Challenge

Mice were challenged orally with 10\textsuperscript{6} CFU of wild-type
invasive \emph{Salmonella} Typhimurium strain UK-1 at day 13 of DSS
treatment. At 3 days after UK-1 challenge, mice were sacrificed and
their spleens, Peyer's Patches and mesenteric lymph nodes were isolated
to determine \emph{Salmonella} Typhimurium strain UK-1 CFU. Tissue was
homogenized and serially diluted. Serial dilutions were plated onto XLD
agar plates with a selective growth medium for Salmonella (BD
Biosciences, Franklin Lakes, NJ, USA). Red colonies with a black center
were determined as \emph{Salmonella} Typhimurium strain UK-1.

\textbf{Gene expression}

BMDM, THP-1 cells, or human monocytes were cultured with 200 ng/ml LPS
for 4 hours and then treated with 10 or 100 \selectlanguage{greek}μ\selectlanguage{english}M LMT503 for 18 hours at
37\selectlanguage{ngerman}°C. To investigate gene expression in colon macrophages, mononuclear
cells were isolated from whole colon tissue as described previously (Kim
et al., 2018). Macrophages were further purified with MagniSort Mouse
F4/80 Positive Selection Kit (Invitrogen, Carlsbad, CA, USA). Whole RNAs
were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA),
followed by cDNA synthesis using a SuperScript II Reverse Transcriptase
(Invitrogen, Carlsbad, CA, USA). Gene expression was measured by
StepOnePlus RT-PCR (Applied Biosystems, Waltham, MA, UNA). Primers used
for PCR are listed in Supplementary Table 1. Each sample was run in
triplicate and three independent experiments were performed for each
sample.

\textbf{Histology}

Colon tissues fixed in neutral buffered formalin were trimmed,
processed, and embedded in paraffin. Then 5-\selectlanguage{greek}µ\selectlanguage{english}m thick sliced tissues of
the paraffin block were stained by hematoxylin and eosin (H\&E). H\&E
stained colonic tissue sections were then scored according to the
following semi-quantitative measurement system for three parameters: 1)
severity of inflammation (0 = rare inflammatory cells in the lamina
propria; 1 = increased numbers of granulocytes in the lamina propria; 2
= confluence of inflammatory cells extending into the submucosa; and 3 =
transmural extension of the inflammatory infiltrate); 2) crypt damage (0
= intact crypts; 1 = loss of the basal one third; 2 = loss of the basal
two thirds; 3 = entire crypt loss; 4 = change of epithelial surface with
erosion; and 5 = confluent erosion); and 3) ulceration (0 = absence of
ulcer; 1 = 1 or 2 foci of ulcerations; 2 = 3 or 4 foci of ulcerations;
and 3 = confluent or extensive ulceration). Values from these three
parameters were added to give a maximal histological score of 11.

\textbf{Cytokine analysis}

Whole colon tissues were homogenized using a tissue homogenizer (Minilys
personal homogenizer, Bertin). Homogenates were analyzed for pro- and
anti-inflammatory cytokines. TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-6, MCP-1, and IL-10 were
analyzed by flow cytometry using a BD Cytometric Beads Assay Mouse
inflammation kit (BD Biosciences, Franklin Lakes, NJ, USA). IL-1\selectlanguage{greek}β \selectlanguage{english}and
IL-22 levels were detected using a mouse IL-1\selectlanguage{greek}β \selectlanguage{english}ELISA Kit (Invitrogen,
Carlsbad, CA, USA) and a mouse IL-22 ELISA Kit (Invitrogen, Carlsbad,
CA, USA), respectively, following the manufacturer's instructions.

\textbf{Statistics}

All statistical analyses were performed using Graphpad Prism version 9
software (GraphPad Software, LLC, San Diego, CA, USA). An unpaired
two-tailed Student's t-test was used to compare differences between two
groups when data had a Gaussian distribution with similar variances. To
compare multiple groups, one-way analysis of variance (ANOVA) was
performed followed by Tukey's post hoc test. Values of
\emph{p}\textless{} 0.05 were considered significant.

\textbf{Results}

\textbf{NAD\textsuperscript{+} enhancer modulates macrophage phenotype
into an anti-inflammatory character}

LMT503, a novel NAD\textsuperscript{+} enhancer, was discovered by Lmito
Therapeutics in an attempt to find IBD therapeutic candidates via
modulation of macrophages (Figure 1a). NAD\textsuperscript{+} synthesis
in macrophages has been reported to contribute to immune suppressive
functions by increasing oxidative phosphorylation (Minhas et al., 2019).
LMT503 works as an efficient substrate for NQO1 which can increase
cellular NAD\textsuperscript{+}/NADH ratio in BMDMs (Figure 1b). To
investigate the effect of LMT503 on inflammatory macrophages, BMDMs were
stimulated with LPS and then treated with LMT503. LMT503 treatment
decreased levels of inflammatory signature molecules TNF-\selectlanguage{greek}α \selectlanguage{english}and iNOS
(Figure 1c) but increased anti-inflammatory signature molecules Arg1 and
IL-10 (Figure 1d) in BMDMs. Sirtuins, a class III histone deacetylases
dependent on NAD\textsuperscript{+}, can inhibit NLRP3-inflammatory
pathways of macrophages and favor anti-inflammatory polarization of
macrophages (Pan, Dong, Huang, \& Fang, 2022; S. Y. Park et al., 2017).
LMT503 treated BMDMs showed increased expression of Sirtuins 1, 3, and 6
(Figure 4e). Upregulated expression of GATA3 is known to induce
anti-inflammatory polarization of macrophages (Faas et al., 2021).
Treatment with LMT503 increased the expression of GATA3 in BMDMs (Figure
1f). However, pro-inflammatory cytokine IL-1\selectlanguage{greek}β \selectlanguage{english}level was decreased after
LMT503 treatment (Figure 1g). Human monocytic THP-1 cells and human
monocytes also showed increased levels of anti-inflammatory markers Arg1
and IL-10 (Figure 1h,i). These data suggest that LMT503 treatment could
induce shift of pro-inflammatory macrophages to an anti-inflammatory
character.

\textbf{NAD\textsuperscript{+} enhancer ameliorates DSS-induced colitis
in mice}

To investigate the anti-inflammatory effect of LMT503 on IBD \emph{in
vivo} , mouse DSS-induced colitis model was used in this study. Mice
received DSS for 5 days followed by oral treatment with LMT503 or
tofacitinib from day 5 when DSS was replaced by normal drinking water.
LMT503 treated mice showed alleviated body weight loss, which was
comparable to an IBD therapeutic JAK-inhibitor tofacitinib (Figure 2a)
(Sandborn et al., 2014). LMT503 treated mice also showed reduced disease
activity index (Figure 2b), recovered colon length (Figure 2c), and
bacterial translocation in mesenteric lymph nodes compared to mice in
the vehicle control group (Figure 2d). H\&E staining of colon tissues
showed reduced inflammation in LMT503 treated groups (Figure 2e).
Pro-inflammatory cytokines TNF, MCP-1, and IL-1\selectlanguage{greek}β \selectlanguage{english}were also significantly
reduced in LMT503 treated groups (Figure 2f).

To further evaluate inflammation in colon tissues, innate immune cell
infiltration was analyzed. Percentage and absolute numbers of
CD11b\textsuperscript{+}Ly6G\textsuperscript{+} neutrophils and
CD11b\textsuperscript{+}Ly6C\textsuperscript{+} monocytes were decreased
significantly in LMT503 treated groups (Figure 3a-d). However,
macrophage and dendritic cell percentage did not differ between LMT503
treated groups and vehicle control groups (Figures 3e,f and Figure
S1a,b).

\textbf{NAD\textsuperscript{+} enhancer modulates mouse colon macrophage
shift to an anti-inflammatory character}

During colonic inflammation, neutrophils and monocytes can be
infiltrated into the colon, although they exist at a low level in steady
state. These inflammatory immune cells can be summoned by tissue
resident immune competent cells such as macrophages. We further analyzed
colonic macrophages in mouse DSS-induced colitis model. Treatment with
LMT503 did not alter the percentage or the number of pro-inflammatory
CX\textsubscript{3}CR1\textsuperscript{int}CD206\textsuperscript{low} or
anti-inflammatory
CX\textsubscript{3}CR1\textsuperscript{hi}CD206\textsuperscript{hi}populations
(Figure 4a-c). However, TNF-\selectlanguage{greek}α \selectlanguage{english}and iNOS levels were decreased while IL-10
levels were increased following LMT503 treatment in colon and small
intestinal macrophages (Figures 4d,e and Figure S1d). Levels of
chemokines such as MCP-1/CCL2, KC/CXCL1, and MIP2/CXCL2 were decreased
after LMT503 treatment in colon macrophages (Figure 4f), suggesting that
colon CX\textsubscript{3}CR1\textsuperscript{+}macrophages contributed
to reduced infiltration of neutrophils and monocytes to the intestine
(Figure 3a-d). These data suggest that colon
CX\textsubscript{3}CR1\textsuperscript{+} macrophage population was not
altered after LMT503 treatment, although their character shifted to an
anti-inflammatory character to ameliorate DSS-induced colitis.

LMT503 treatment altered macrophages to an anti-inflammatory character
to alleviate DSS-induced colitis. The anti-inflammatory environment
might arouse susceptibility to infection. To investigate this issue,
mice treated with LMT503 were challenged with
\emph{Salmonella}Typhimurium strain UK-1 at day 13 at the recovery phase
of DSS-induced colitis. LMT503 treated group did not show significant
body weight loss compared to the vehicle control group (Figure 5a).
\emph{Salmonella}Typhimurium trafficking to the MLN and spleen did not
differ between vehicle control and LMT503 treated groups (Figure 5b). At
the recovery phase of colitis, host defense including production of
pro-inflammatory cytokines TNF, IL-6, and MCP-1 levels in the colon
tissue did not differ significantly after LMT503 treatment (Figure 5c).
These data suggest that LMT503 treatment did not increase vulnerability
to bacterial infections.

\textbf{Colonic CX\textsubscript{3}CR1\textsuperscript{+} macrophages
play a crucial role in the anti-inflammatory effect of LMT503 in
DSS-induced colitis}

Although the ratio and absolute numbers of colon macrophage populations
were not altered, LMT503 treatment resulted in polarization from
inflammatory to an anti-inflammatory character. It is known that colon
resident macrophages show high expression of CX\textsubscript{3}CR1.
During colitis, Ly6C\textsuperscript{+} monocyte infiltration to the
lamina propria can differentiate into
CX\textsubscript{3}CR1\textsuperscript{int} macrophages which are
pro-inflammatory. However, afterwards, further maturation into
CX\textsubscript{3}CR1\textsuperscript{hi} regulatory macrophages drives
resolution of inflammation (Bain et al., 2013). These gut
CX\textsubscript{3}CR1\textsuperscript{hi} macrophages can clear out
apoptotic neutrophils and support tissue healing (Arandjelovic \&
Ravichandran, 2015; Castoldi et al., 2023). Thus,
CX\textsubscript{3}CR1\textsuperscript{+} macrophages are crucial in
maintaining gut homeostasis. They play a key role in IBD. To verify that
macrophage character shift by LMT503 treatment contributed to
DSS-induced colitis alleviation, we used a
CX\textsubscript{3}CR1\textsuperscript{+} macrophage depletion system.
For depletion of CX\textsubscript{3}CR1\textsuperscript{+} cells,
CX\textsubscript{3}CR1-DTR mice were \emph{i.p.} injected with
diphtheria toxin during DSS-induced colitis. Under DSS treatment, mice
deficient of CX\textsubscript{3}CR1\textsuperscript{+} macrophages
showed body weight loss and high score of disease activity index,
similar to those of WT mice (Figure 6a,b). However, absence of
CX\textsubscript{3}CR1\textsuperscript{+} macrophages abrogated
alleviation of colitis including less body weight loss, low score of
disease activity index, and recovered colon length after LMT503
treatment. At day 10, group comparison of body weight changes, disease
activity index, and colon length suggested that the effect of LMT503
treatment could be abolished in absence of
CX\textsubscript{3}CR1\textsuperscript{+} macrophages (Figure 6c-e).
After CX\textsubscript{3}CR1\textsuperscript{+} macrophage depletion,
LMT503 could not control pro-inflammatory cytokines IL-6 (Figure 6f).
LMT503 treatment significantly increased IL-10 and IL-22 production in
inflamed colon which could be diminished after depletion of
CX\textsubscript{3}CR1\textsuperscript{+} macrophages (Figure 6g). These
data suggest that LMT503 can exert its effect through targeted
modulation of CX\textsubscript{3}CR1\textsuperscript{+} macrophages to
alleviate DSS-induced colitis.

\textbf{Discussion}

Intestinal macrophages are known as ``gatekeepers'' for their ability to
clear foreign pathogens by expressing pathogen-associated molecular
patterns (PAMPs) and recognize tissue damage by damage-associated
molecular patterns (DAMPs). They also show tolerance to food or
commensal microbes. Macrophages are a heterogeneous population of innate
immune cells that play diverse functions in immune response and tissue
homeostasis. Although boundaries of the heterogeneous macrophage
population are ambiguous, they can be functionally divided into
pro-inflammatory versus anti-inflammatory macrophages. Macrophage
polarization depends on their microenvironment. Disruption of the
composition or function of macrophages can contribute to various
gastro-intestinal diseases such as IBD. IBD patients show an altered
population of macrophages where inflammatory macrophages are dominant.
These inflammatory macrophages can accumulate in the intestine during
IBD, which can release TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6. To overcome IBD,
biologics such as anti-TNF therapy is commonly used since TNF-\selectlanguage{greek}α \selectlanguage{english}is a key
pathological cytokine in IBD. Anti-IL-12/23, anti-integrin,
5-aminosalicylic acids, and immunomodulators can also be used for IBD
therapy. Despite recent advances in IBD therapy, limitations still exist
since complete remission is difficult. Thus, the goal for overcoming IBD
is shifting to targeting mucosal healing.

Since macrophages are deeply involved in regulating inflammation and
mucosal healing, targeting these macrophages can be a good therapeutic
strategy against IBD. Many previous studies targeting macrophages to
induce an anti-inflammatory shift have shown the potential of an
effective IBD therapy. Activation of SIRT1 can promote anti-inflammatory
genes Mrc1 and IL-10 while decreasing pro-inflammatory genes iNOS, CCL2,
IL-12 p35, and IL-12 p40. Anti-inflammatory macrophage polarization by
the SIRT1/adenosine monophosphate-activated protein kinase pathway can
reduce inflammatory responses in a rheumatoid arthritis mouse model (S.
Park et al., 2020; Zhou, Zhang, \& Ding, 2022). TLR2/1 agonist PAM3CSK4
can induce monocytes to differentiate into anti-inflammatory macrophages
and reduce DSS-induced colitis in a mouse model (Horuluoglu,
Kayraklioglu, Tross, \& Klinman, 2020).

Metabolic pathways in macrophages are deeply involved in the fate of
macrophage polarization. Therefore, reports targeting macrophage
polarization to an anti-inflammatory character by metabolic modulation
is arising as a promising strategy for IBD therapy. Pro-inflammatory
macrophages depend on glycolysis, while anti-inflammatory macrophages
are activated by mitochondrial oxidative phosphorylation (Liu et al.,
2021). Different paths of arginine catabolism contribute to macrophage
polarization. Generation of NO by iNOS favors inflammatory macrophage
polarization, while generation of urea and orthinine by Arg1 favors
anti-inflammatory macrophage polarization. Increased consumption of
L-arginine by iNOS and decreased Arg1 have been shown in ulcerative
colitis. Thus, dietary supplementation of L-arginine can be used as an
IBD therapy (Andrade et al., 2018; Bourgonje et al., 2020).

LMT503 is an NAD\textsuperscript{+} enhancer by working as an efficient
substrate for NQO1. NQO1 has multiple roles in defending against
cellular stress. NQO1 can detoxify quinones by reducing them into
hydroquinones. At the same time, oxidation of NAD(P)H occurs, which
increases NAD\textsuperscript{+}. Previous studies have shown that
inflammation can be controlled by triggering macrophage shift by
increasing NAD\textsuperscript{+} level. NAD\textsuperscript{+}synthesis
via the kynurenine pathway can shift aged inflammatory macrophages to an
anti-inflammatory state with increased phagocytic activity (Minhas et
al., 2019). Increasing NAD\textsuperscript{+} by administration of
NAD\textsuperscript{+} precursor \selectlanguage{greek}β-\selectlanguage{english}nicotinamide mononucleotide (\selectlanguage{greek}β-\selectlanguage{english}NMN)
can shift macrophages to an anti-inflammatory type during sepsis (Cros
et al., 2022). Sirtuins are a family of class III histone deacetylases
known to be dependent on NAD\textsuperscript{+}(Anderson, Madsen, Olsen,
\& Hirschey, 2017). They consist of seven members, SIRT1-7. Sirtuins can
modulate inflammation via nuclear factor-kappa B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) or activator
protein 1 (AP-1). SIRTs except SIRT5 are known to inhibit NF-\selectlanguage{greek}κ\selectlanguage{english}B
activation by deacetylation of p65 subunit. SIRT1 cam inhibit IL-1\selectlanguage{greek}β
\selectlanguage{english}secretion by down-regulating NLRP3 inflammasome (S. Park et al., 2020).
SIRT1, SIRT3, and SIRT6 can decrease AP-1 transcriptional activity which
controls pro-inflammatory cytokine production (Palomer et al., 2020;
Zhang et al., 2010). Treatment of BMDMs with LMT503 increased SIRT1, 3,
and 6.

In summary, this study found that NAD\textsuperscript{+} enhancement
alleviated intestinal inflammation by reinforcing immune regulatory
character of tissue resident
CX\textsubscript{3}CR1\textsuperscript{+}macrophages.
NAD\textsuperscript{+} enhancement was achieved by LMT503, an efficient
substrate for NQO1. Metabolic reprograming in macrophages treated with
LMT503 down-regulated inflammatory markers TNF-\selectlanguage{greek}α \selectlanguage{english}and iNOS but
up-regulated anti-inflammatory IL-10 and Arg1 during colitis.
NAD\textsuperscript{+} enhancement of colonic
CX\textsubscript{3}CR1\textsuperscript{+} macrophages was crucial for
ameliorating DSS-induced colitis in mice since LMT503 treatment did not
show any effect when
CX\textsubscript{3}CR1\textsuperscript{+}macrophages were depleted. Our
study suggests that LMT503 is a potential IBD therapy candidate that can
drive anti-inflammatory macrophage polarization via immuno-metabolic
modulation.

\textbf{Author Contributions}

Young-In Kim, Yong Rae Hong, Wheeseong Lee, and Sun-Young Chang
conceived and designed experiments. Young-In Kim, Inseok Ko, Eun-Je Yi,
and Jusik Kim performed experiments and analysis. Inseok Ko and
Wheeseong Lee provided critical materials. Young-In Kim and Sun-Young
Chang wrote the manuscript and provided creative input.

\textbf{Acknowledgements}

This work was supported by Korea Initiative for fostering University of
Research and Innovation Program of the National Research Foundation
(NRF) funded by the Korean government (MSIT) (NRF-2021M3H1A104892211,
NRF-2022R1I1A1A01069464, NRF-2023R1A2C1003557). Graphics were created
with BioRender.com.

\textbf{Conflict of Interest}

The authors declare no conflicts of interest.



\textbf{Figure Legends}

Figure 1. NAD\textsuperscript{+} enhancement shifts macrophage
polarization into an anti-inflammatory character.

(a) LMT503 structure. (b-g) BMDM originated from wild-type B6 mice were
treated with 200 ng/ml LPS for 4 h followed by treatment with 10 \selectlanguage{greek}μ\selectlanguage{english}M or
100 \selectlanguage{greek}μ\selectlanguage{english}M LMT503 for 18 h. (b) NQO1 activity measured by OD change per min.
Gene expression of (c) inflammatory markers, (d) anti-inflammatory
markers, (e) Sirtuins 1, 3, 6, and (f) GATA3 in BMDMs. (g) IL-1\selectlanguage{greek}β
\selectlanguage{english}expression in culture supernatant of BMDMs stimulated with 200 ng/ml LPS
3 h followed by 5 mM ATP 1h, afterwards 10 \selectlanguage{greek}μ\selectlanguage{english}M or 100 \selectlanguage{greek}μ\selectlanguage{english}M LMT503 for 18 h.
(h) Human monocyte THP-1 cells or (i) human peripheral blood derived
monocytes were treated with 200 ng/ml LPS 4 h followed by treatment with
10 \selectlanguage{greek}μ\selectlanguage{english}M LMT530 for 18 h. ns; not significant, *, \emph{p} \textless{}
0.05, **, \emph{p} \textless{} 0.01, ***, \emph{p} \textless{} 0.001,
one-way ANOVA with multiple comparison.

Figure 2. NAD\textsuperscript{+} enhancement ameliorates inflammation in
a murine DSS-induced colitis model.

C57BL/6 female mice were given 2\% DSS in drinking water for 5 days.
Mice were then provided with normal drinking water afterwards (n =
9/group, n = 5 for healthy control). After DSS treatment, 50 mg/kg
LMT503 or 30 mg/kg tofacitinib was then orally given to mice every day
starting from Day 5.

(a) Body weight, (b) disease activity index (***, \emph{p} \textless{}
0.001 one-way ANOVA with multiple comparison vs. vehicle control group),
(c) colon length, and (d) bacterial translocation (CFU/g) in the MLN.

(e) H\&E staining of mouse colon and histological score.

(f) Cytokine levels (TNF, MCP-1, and IL-1\selectlanguage{greek}β\selectlanguage{english}) of colon homogenates.
*p\textless{}0.05, **p\textless{}0.01, ***p\textless{}0.001 one-way
ANOVA with multiple comparison.

Figure 3. NAD\textsuperscript{+} enhancement reduces infiltration of
innate inflammatory cells in colon of DSS-induced colitis mouse.

Percentage and absolute number of (a, b)
Ly6G\textsuperscript{+}CD11b\textsuperscript{+} neutrophils, (c, d)
Ly6C\textsuperscript{+}CD11b\textsuperscript{+} monocytes, and (e, f)
F4/80\textsuperscript{+}CD11b\textsuperscript{+} macrophages from colon
lamina propria were analyzed (n = 5/group). ns, not significant;
*\emph{p} \textless{} 0.05, **\emph{p} \textless{} 0.01,
***\emph{p}\textless{} 0.001 one-way ANOVA with multiple comparison.

Figure 4. Metabolic reprograming of colon macrophages shift to an
anti-inflammatory character.

C57BL/6 female mice were given 2\% DSS in drinking water for 5 days.
Mice were then provided with normal drinking water (n = 6/group). Mice
were given 50 mg/kg LMT503 orally everyday starting from Day 5. At day
10, macrophages isolated from colon lamina propria were analyzed for
sub-population and gene expression.

(a) Colon macrophage population. CD206 and CX\textsubscript{3}CR1 were
analyzed among CD11b\textsuperscript{+} gated cells.

(b) CX\textsubscript{3}CR1\textsuperscript{hi} cell population and cell
number among CD11b\textsuperscript{+} gated cells.

(c) CX\textsubscript{3}CR1\textsuperscript{int} cell population and cell
number among CD11b\textsuperscript{+} gated cells.

(d) Inflammatory cytokines, (e) anti-inflammatory cytokines, and (f)
chemokine expression in colon macrophages. ns, not significant,
*\emph{p} \textless{} 0.05, **\emph{p \textless{}} 0.01,
***\emph{p}\textless{} 0.001 one-way ANOVA with multiple comparison.

Figure 5. Treatment with LMT503 does not increase vulnerability
to\emph{Salmonella} Typhimurium infection. At day 13 of DSS-induced
colitis, mice (n = 6/group) were infected orally with
10\textsuperscript{6} CFU of \emph{Salmonella} Typhimurium (UK-1).
Bacteria count (CFU) and cytokine levels in colon tissues were analyzed
at 3 days after infection.

(a) Body weight, (b) UK-1 (CFU/g) in the spleen, Peyer's Patches, and
mesenteric lymph nodes. (c) Cytokine levels in colon homogenates.

Figure 6. CX\textsubscript{3}CR1\textsuperscript{+} macrophages are
critical for immune regulation via metabolic reprograming.

For macrophage depletion, CX\textsubscript{3}CR1-DTR mice
were\emph{i.p.} injected with 200 ng diphtheria toxin at Days 1, 4, and
7. At day 10, colon length and cytokines were analyzed (n = 6/group).

(a) Body weight, (b) disease activity index (*** \emph{p} \textless{}
0.001, two-way ANOVA with multiple comparison), (c) body weight at day
10, (d) disease activity index at day 10, (e) colon length, and (f, g)
cytokine gene expressions of colon homogenates. ns, not significant;
*\emph{p} \textless{}0.05, **\emph{p} \textless{}0.01, ***\emph{p}
\textless{}0.001 one-way ANOVA with multiple comparison.

\textbf{References}

Anderson, K. A., Madsen, A. S., Olsen, C. A., \& Hirschey, M. D. (2017).
Metabolic control by sirtuins and other enzymes that sense NAD(+), NADH,
or their ratio. \emph{Biochim Biophys Acta Bioenerg, 1858} (12),
991-998. doi:10.1016/j.bbabio.2017.09.005

Andrade, W. B., Jacinto, J. L., da Silva, D. K., Roveratti, M. C.,
Estoche, J. M., Oliveira, D. B., \ldots{} Aguiar, A. F. (2018).
l-Arginine supplementation does not improve muscle function during
recovery from resistance exercise. \emph{Appl Physiol Nutr Metab, 43}
(9), 928-936. doi:10.1139/apnm-2017-0594

Arandjelovic, S., \& Ravichandran, K. S. (2015). Phagocytosis of
apoptotic cells in homeostasis. \emph{Nat Immunol, 16} (9), 907-917.
doi:10.1038/ni.3253

Bain, C. C., Bravo-Blas, A., Scott, C. L., Perdiguero, E. G., Geissmann,
F., Henri, S., \ldots{} Mowat, A. M. (2014). Constant replenishment from
circulating monocytes maintains the macrophage pool in the intestine of
adult mice. \emph{Nat Immunol, 15} (10), 929-937. doi:10.1038/ni.2967

Bain, C. C., Scott, C. L., Uronen-Hansson, H., Gudjonsson, S., Jansson,
O., Grip, O., \ldots{} Mowat, A. M. (2013). Resident and
pro-inflammatory macrophages in the colon represent alternative
context-dependent fates of the same Ly6Chi monocyte
precursors.\emph{Mucosal Immunol, 6} (3), 498-510.
doi:10.1038/mi.2012.89

Bourgonje, A. R., Feelisch, M., Faber, K. N., Pasch, A., Dijkstra, G.,
\& van Goor, H. (2020). Oxidative Stress and Redox-Modulating
Therapeutics in Inflammatory Bowel Disease. \emph{Trends Mol Med, 26}
(11), 1034-1046. doi:10.1016/j.molmed.2020.06.006

Castoldi, A., Sanin, D. E., van Teijlingen Bakker, N., Aguiar, C. F., de
Brito Monteiro, L., Rana, N., \ldots{} Saraiva Camara, N. O. (2023).
Metabolic and functional remodeling of colonic macrophages in response
to high-fat diet-induced obesity. \emph{iScience, 26} (10), 107719.
doi:10.1016/j.isci.2023.107719

Cros, C., Margier, M., Cannelle, H., Charmetant, J., Hulo, N., Laganier,
L., \ldots{} Canault, M. (2022). Nicotinamide Mononucleotide
Administration Triggers Macrophages Reprogramming and Alleviates
Inflammation During Sepsis Induced by Experimental
Peritonitis.\emph{Front Mol Biosci, 9} , 895028.
doi:10.3389/fmolb.2022.895028

Diehl, G. E., Longman, R. S., Zhang, J. X., Breart, B., Galan, C.,
Cuesta, A., \ldots{} Littman, D. R. (2013). Microbiota restricts
trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi)
cells.\emph{Nature, 494} (7435), 116-120. doi:10.1038/nature11809

Faas, M., Ipseiz, N., Ackermann, J., Culemann, S., Gruneboom, A.,
Schroder, F., \ldots{} Kronke, G. (2021). IL-33-induced metabolic
reprogramming controls the differentiation of alternatively activated
macrophages and the resolution of inflammation. \emph{Immunity, 54}
(11), 2531-2546 e2535. doi:10.1016/j.immuni.2021.09.010

Geissmann, F., Jung, S., \& Littman, D. R. (2003). Blood monocytes
consist of two principal subsets with distinct migratory
properties.\emph{Immunity, 19} (1), 71-82.
doi:10.1016/s1074-7613(03)00174-2

Harber, K. J., de Goede, K. E., Verberk, S. G. S., Meinster, E., de
Vries, H. E., van Weeghel, M., \ldots{} Van den Bossche, J. (2020).
Succinate Is an Inflammation-Induced Immunoregulatory Metabolite in
Macrophages. \emph{Metabolites, 10} (9)doi:10.3390/metabo10090372

Hill, D. A., \& Artis, D. (2010). Intestinal bacteria and the regulation
of immune cell homeostasis. \emph{Annu Rev Immunol, 28} , 623-667.
doi:10.1146/annurev-immunol-030409-101330

Horuluoglu, B. H., Kayraklioglu, N., Tross, D., \& Klinman, D. (2020).
PAM3 protects against DSS-induced colitis by altering the M2:M1
ratio.\emph{Sci Rep, 10} (1), 6078. doi:10.1038/s41598-020-63143-z

Kaser, A., Zeissig, S., \& Blumberg, R. S. (2010). Inflammatory bowel
disease. \emph{Annu Rev Immunol, 28} , 573-621.
doi:10.1146/annurev-immunol-030409-101225

Kim, Y. I., Song, J. H., Ko, H. J., Kweon, M. N., Kang, C. Y.,
Reinecker, H. C., \& Chang, S. Y. (2018). CX(3)CR1(+) Macrophages and
CD8(+) T Cells Control Intestinal IgA Production. \emph{J Immunol, 201}
(4), 1287-1294. doi:10.4049/jimmunol.1701459

Kim, Y. I., Yi, E. J., Kim, Y. D., Lee, A. R., Chung, J., Ha, H. C.,
\ldots{} Chang, S. Y. (2020). Local Stabilization of Hypoxia-Inducible
Factor-1alpha Controls Intestinal Inflammation via Enhanced Gut Barrier
Function and Immune Regulation. \emph{Front Immunol, 11} , 609689.
doi:10.3389/fimmu.2020.609689

Lin, S. L., Li, B., Rao, S., Yeo, E. J., Hudson, T. E., Nowlin, B. T.,
\ldots{} Duffield, J. S. (2010). Macrophage Wnt7b is critical for kidney
repair and regeneration. \emph{Proc Natl Acad Sci U S A, 107} (9),
4194-4199. doi:10.1073/pnas.0912228107

Liu, Y., Xu, R., Gu, H., Zhang, E., Qu, J., Cao, W., \ldots{} Cai, Z.
(2021). Metabolic reprogramming in macrophage responses. \emph{Biomark
Res, 9} (1), 1. doi:10.1186/s40364-020-00251-y

Maloy, K. J., \& Powrie, F. (2011). Intestinal homeostasis and its
breakdown in inflammatory bowel disease. \emph{Nature, 474} (7351),
298-306. doi:10.1038/nature10208

Mazzini, E., Massimiliano, L., Penna, G., \& Rescigno, M. (2014). Oral
tolerance can be established via gap junction transfer of fed antigens
from CX3CR1(+) macrophages to CD103(+) dendritic cells. \emph{Immunity,
40} (2), 248-261. doi:10.1016/j.immuni.2013.12.012

Minhas, P. S., Liu, L., Moon, P. K., Joshi, A. U., Dove, C., Mhatre, S.,
\ldots{} Andreasson, K. I. (2019). Macrophage de novo NAD(+) synthesis
specifies immune function in aging and inflammation. \emph{Nat Immunol,
20} (1), 50-63. doi:10.1038/s41590-018-0255-3

Murray, P. J., \& Wynn, T. A. (2011). Protective and pathogenic
functions of macrophage subsets. \emph{Nat Rev Immunol, 11} (11),
723-737. doi:10.1038/nri3073

Na, Y. R., Stakenborg, M., Seok, S. H., \& Matteoli, G. (2019).
Macrophages in intestinal inflammation and resolution: a potential
therapeutic target in IBD. \emph{Nat Rev Gastroenterol Hepatol, 16} (9),
531-543. doi:10.1038/s41575-019-0172-4

Nebert, D. W., Roe, A. L., Vandale, S. E., Bingham, E., \& Oakley, G. G.
(2002). NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to
benzene, and predisposition to disease: a HuGE review. \emph{Genet Med,
4} (2), 62-70. doi:10.1097/00125817-200203000-00003

Palmieri, E. M., Gonzalez-Cotto, M., Baseler, W. A., Davies, L. C.,
Ghesquiere, B., Maio, N., \ldots{} McVicar, D. W. (2020). Nitric oxide
orchestrates metabolic rewiring in M1 macrophages by targeting aconitase
2 and pyruvate dehydrogenase. \emph{Nat Commun, 11} (1), 698.
doi:10.1038/s41467-020-14433-7

Palomer, X., Roman-Azcona, M. S., Pizarro-Delgado, J., Planavila, A.,
Villarroya, F., Valenzuela-Alcaraz, B., \ldots{} Vazquez-Carrera, M.
(2020). SIRT3-mediated inhibition of FOS through histone H3
deacetylation prevents cardiac fibrosis and inflammation. \emph{Signal
Transduct Target Ther, 5} (1), 14. doi:10.1038/s41392-020-0114-1

Pan, Z., Dong, H., Huang, N., \& Fang, J. (2022). Oxidative stress and
inflammation regulation of sirtuins: New insights into common oral
diseases. \emph{Front Physiol, 13} , 953078.
doi:10.3389/fphys.2022.953078

Park, S., Shin, J., Bae, J., Han, D., Park, S. R., Shin, J., \ldots{}
Park, H. W. (2020). SIRT1 Alleviates LPS-Induced IL-1beta Production by
Suppressing NLRP3 Inflammasome Activation and ROS Production in
Trophoblasts. \emph{Cells, 9} (3)doi:10.3390/cells9030728

Park, S. Y., Lee, S. W., Lee, S. Y., Hong, K. W., Bae, S. S., Kim, K.,
\& Kim, C. D. (2017). SIRT1/Adenosine Monophosphate-Activated Protein
Kinase alpha Signaling Enhances Macrophage Polarization to an
Anti-inflammatory Phenotype in Rheumatoid Arthritis. \emph{Front
Immunol, 8} , 1135. doi:10.3389/fimmu.2017.01135

Podolsky, D. K. (1991). Inflammatory bowel disease (1). \emph{N Engl J
Med, 325} (13), 928-937. doi:10.1056/NEJM199109263251306

Rath, M., Muller, I., Kropf, P., Closs, E. I., \& Munder, M. (2014).
Metabolism via Arginase or Nitric Oxide Synthase: Two Competing Arginine
Pathways in Macrophages. \emph{Front Immunol, 5} , 532.
doi:10.3389/fimmu.2014.00532

Ross, D., \& Siegel, D. (2021). The diverse functionality of NQO1 and
its roles in redox control. \emph{Redox Biol, 41} , 101950.
doi:10.1016/j.redox.2021.101950

Russell, D. G., Huang, L., \& VanderVen, B. C. (2019). Immunometabolism
at the interface between macrophages and pathogens. \emph{Nat Rev
Immunol, 19} (5), 291-304. doi:10.1038/s41577-019-0124-9

Sandborn, W. J., Ghosh, S., Panes, J., Vranic, I., Wang, W.,
Niezychowski, W., \& Study, A. I. (2014). A phase 2 study of
tofacitinib, an oral Janus kinase inhibitor, in patients with Crohn's
disease. \emph{Clin Gastroenterol Hepatol, 12} (9), 1485-1493 e1482.
doi:10.1016/j.cgh.2014.01.029

Viola, M. F., \& Boeckxstaens, G. (2021). Niche-specific functional
heterogeneity of intestinal resident macrophages. \emph{Gut, 70} (7),
1383-1395. doi:10.1136/gutjnl-2020-323121

Wculek, S. K., Heras-Murillo, I., Mastrangelo, A., Mananes, D., Galan,
M., Miguel, V., \ldots{} Sancho, D. (2023). Oxidative phosphorylation
selectively orchestrates tissue macrophage homeostasis. \emph{Immunity,
56} (3), 516-530 e519. doi:10.1016/j.immuni.2023.01.011

Wynn, T. A., \& Vannella, K. M. (2016). Macrophages in Tissue Repair,
Regeneration, and Fibrosis. \emph{Immunity, 44} (3), 450-462.
doi:10.1016/j.immuni.2016.02.015

Zhang, R., Chen, H. Z., Liu, J. J., Jia, Y. Y., Zhang, Z. Q., Yang, R.
F., \ldots{} Liang, C. C. (2010). SIRT1 suppresses activator protein-1
transcriptional activity and cyclooxygenase-2 expression in
macrophages.\emph{J Biol Chem, 285} (10), 7097-7110.
doi:10.1074/jbc.M109.038604

Zhou, Y., Zhang, F., \& Ding, J. (2022). As a Modulator, Multitasking
Roles of SIRT1 in Respiratory Diseases. \emph{Immune Netw, 22} (3), e21.
doi:10.4110/in.2022.22.e21

\selectlanguage{english}
\FloatBarrier
\end{document}

