Table Of ContentMethods in
Molecular Biology 2184
Suresh Mishra Editor
Immuno-
metabolism
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
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Immunometabolism
Methods and Protocols
Edited by
Suresh Mishra
Faculty of Health Sciences, Department of Internal Medicine, University of Manitoba, Winnipeg, MB,
Canada; Faculty of Health Sciences, Department of Physiology and Pathophysiology, University of
Manitoba, Winnipeg, MB, Canada
Editor
SureshMishra
FacultyofHealthSciences
DepartmentofInternalMedicine
UniversityofManitoba
Winnipeg,MB,Canada
FacultyofHealthSciences
DepartmentofPhysiologyandPathophysiology
UniversityofManitoba
Winnipeg,MB,Canada
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-0801-2 ISBN978-1-0716-0802-9 (eBook)
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Preface
Immunometabolismisanemergingfieldofbiomedicalinvestigationattheinterfaceofthe
historicallydistinctdisciplinesofimmunologyandmetabolism[1];itincorporatesboththe
roleofimmunecellsinmetabolichomeostasisinthebodyandtheimpactofinterconnected
metabolicpathwaysonimmunecellfunctions[2,3].Thebirthofthisnewresearchfrontier
can be traced back to the understanding that obesity affects the immune system and
promotes inflammation, also known as meta-inflammation (or alternatively chronic
low-grade inflammation). For more than 50 years, physicians and scientists have observed
a close association between metabolic disorders and systemic inflammation [4]. Studies
dating from the 1960s found that individuals with type 2 diabetes mellitus have higher
circulating concentrations of active complement and acute-phase reactants [5, 6], both of
whichareclassicalmarkersofaninflammatorystate.However,thesiteofinflammationand
theirpathologicalimplicationsremainedobscureuntilabout25yearsago[4].Inthe1990s,
it was reported that the obesity-induced expression of tumor necrosis factor-α (TNF-α)
exists in the adipose tissue of both rodents and humans, and it has been proposed that
TNF-α mediates obesity-related insulin resistance [7]. Subsequently, the transcriptional
evidence for the presence of macrophages in adipose tissue was found [8, 9]. Later, it was
shown that the macrophage levels correlated positively with adiposity, and most of the
TNF-α and other inflammatory molecules were derived from adipose tissue macrophages
[8,9].
Although the initial immunometabolism studies focused on adipose tissue, it is now
evident that the metabolic activation of the immune system is not limited to obesity [4].
Growing evidence suggests that interacting metabolic pathways in immune cells play a
central role in their functional plasticity and have been the focus of intense interest as
therapeutictargetstoharnessthefullpotentialoftheimmunesystem[2,3].Thescientific
communitydoesnotyetfullyunderstandhowandwhyimmunecellscommittoaparticular
metabolicfate,ortheimmunologicalconsequencesofreachingametabolicendpointbyone
pathway versus another. The multilevel interactions between the metabolic and immune
systems suggest pathogenic mechanisms that may underlie many metabolic and immune
diseasesandoffersubstantialtherapeuticpromise[1].Researchonimmunotherapyhasbeen
conductedforoveracentury;nevertheless,thelastdecadehasseenanincreaseininterestin
studying immunotherapy. However, a number of challenges remain due to its limited
effectiveness and treatment-related adverse effects. It is anticipated that incorporating
immunometabolism and manipulating immune cell functions will provide some much-
needed ways to improve the effectiveness of promising immunotherapy, and reduce the
unintendedsideeffects,aswellasimprovethetreatmentandpreventionofawidevarietyof
pathologiesandchronicdiseases.Thus,themolecularunderpinningofimmunometabolism
hasbecomeaprioritytomaximizethetherapeuticefficacyofimmunotherapy.
Thisbookisdedicatedtoshowcasingthetremendouseffortandprogressmadeoverthe
lastfewdecadesindevelopingtechniquesandprotocols,andinutilizingrecenttechnologi-
caladvancesforprobingandmanipulatingadiposeandimmunecells,andsubsequentlytheir
functions and immunometabolic consequences. All chapters are written by experts in their
particularfieldsandcoverawiderangeoftopicsrelatedtothestudyofimmunometabolism
using different experimental approaches in combination with new tools and techniques.
v
vi Preface
Manychaptersinthisprotocolsbookarewrittenusingmacrophagesasamodelimmunecell
type(includingmurineandhumancelllines,aswellasprimarycells)becausemacrophages
are the most prominent cells of the innate immune system that regulate a variety of
inflammatory, host defense, and wound repair processes, and as such have been studied
extensively for cell differentiation, gene regulation, and signal transduction. In addition,
well-establishedproceduresexisttoisolate,culture,andactivatemouseandhumanmacro-
phages.Importantly,anextraordinaryplasticityofmacrophagestotheirsurroundingmicro-
environment makes them a unique therapeutic target for a variety of immunometabolic
diseases. Moreover, protocols using adipocytes, dendritic cells, and T cells as model cell
lines, as well as measurement of glucose metabolism at the systemic level, have also been
included,asitrelatestoimmunometabolism.
The single-cell RNA sequencing (scRNA-seq) allows an unbiased approach for unco-
vering a new level of cellular heterogeneity and dynamics of a diverse biological system,
includingtheimmunesystem,asitenablesacomprehensiveanalysisofthetranscriptomeof
individual cells by next-generation sequencing. Optimization of the technical procedures
performed prior to RNA-seq analysis is imperative to the success of a scRNA-seq experi-
ment. Janilyn Arsenio describes three major experimental procedures: (1) the isolation of
immuneCD8a+Tcellsfromprimarymurinetissue;(2)thegenerationofsingle-cellcDNA
0
libraries using the 10x Genomics Chromium Controller and the Chromium Single Cell 3
Solution;and(3)cDNAlibraryqualitycontrol.Inthisprotocol,CD8a+Tcellsareisolated
from murine spleen tissue, but any cell type of interest can be enriched and used for the
single-cellcDNAlibrarygenerationandsubsequentRNA-seqexperiments.
Cellular metabolism has emerged as a major player in the regulation of the functional
plasticity of different immune cell types. For example, the production of lactate by macro-
phages has been associated with their polarization and function. Baeza-Lehnert et al.
describeimagingprotocolstocharacterizethemetabolismofculturedhumanmacrophages
using a genetically encoded fluorescent sensor specific for lactate. This protocol allows
determining the kinetic parameters of monocarboxylate transporter 4 and lactate produc-
tionatthesingle-celllevel.Theauthorshavealsoprovidedpracticaladviceregardingsensor
expression, imaging, and data analysis. Importantly, the spatiotemporal resolution of this
techniqueisamenabletothestudyof fasteventsatthesingle-celllevelindifferentimmune
cellsandothercelltypes.
Inadditiontothemeasurementofthetranscriptomeandcellularmetabolismatasingle-
cell level, recent developments have enabled a parallel analysis of both the transcript and
protein at a single-cell level by using antibodies conjugated to barcoded oligonucleotides.
These antibodies allow the “i” of protein levels to be presented in nucleotide format,
permitting a sequencing-based detection of both modalities at a single-cell level. Tapio
Lo¨nnberg and colleagues present a simple and reliable method for the conjugation of
oligonucleotides with antibodies and a protocol for their use in single-cell transcriptome
sequencing.Thisprotocoladdressesthesignificantchallengesassociatedwiththebiological
andfunctionalinterpretationofnewlyidentifiedcellpopulationsusingscRNA-seq.
The stable isotope labeling of metabolites is a technique employed to investigate the
movementofametabolitethroughacell’senzymaticmachinery.Theinformationobtained
fromthisprocessallows for thedeterminingoftherelativefluxesofmetabolitesthrougha
biochemical pathway, and the contribution of specific metabolites to the total metabolite
pool. For instance, the incorporation of stable isotope labels into specific fatty acids allows
for the discrimination of newly synthesized fatty acids from those that are in preexisting
pools, or fatty acids that have been imported from an extracellular source. Kevin Williams
Preface vii
and Steven Bensinger describe a workflow for a total cellular fatty acid analysis in macro-
phages, which combines a fatty acid methyl ester analysis (by gas chromatography–mass
spectrometry) with isotopic labeling. This approach can elucidate the synthetic pathways
beingengagedbythecellsandtherelativecontributionofsynthesisandimporttomaintain
lipidcontent,whichisanimportantcomponentofcellular metabolisminimmunecells.
Discovery-basedquantitativeproteomicsisausefulmethodtounravelcomplexprotein
networks and protein-protein interactions. Saiful Chowdhury and coworkers describe pro-
tocolsfortheproteomicsnetworkanalysisofpolarizedmacrophagesinresponsetopro-and
anti-inflammatoryagents.Theyprovidedetailedprotocols,aquantitativeproteomicanalysis
bymassspectrometrydata,aproteinnetworkanalysisbybioinformatics,andavalidationof
targets through biochemical methods (e.g., immunocytochemistry, immunoblotting, gene
silencing,andreal-timePCR).
The intercellular communication or cross talk between different cell types, including
intra-organ and interorgan cross talk, engaged in metabolic and immune regulation (e.g.,
adipocytes, hepatocytes, macrophages, dendritic cells, lymphocytes) plays a crucial role in
immunometabolism at the systemic level and their dysregulation in the development of a
number of metabolic and immune diseases, including different types of cancer. In this
context, exosomes have been identified as a crucial player in the intercellular cross talk in
healthanddisease[10].Thus,itiscrucialtodevelopprotocolstoinvestigatetheintercellu-
lar cross talk between metabolic and immune cells, including the role of exosomes in this
process. To this end, Fridman et al. describe methods for the isolation and polarization of
mouseperitonealmacrophages,thepurificationofexosomesfromtheconditionedmediaof
thepolarizedmacrophages,andthecharacterizationoftheresultingexosomes.Inaddition,
they provide protocols to study exosome-based communication between two cell types
using macrophages and pancreatic cancer cells as an example, thus mimicking a tumor
microenvironment. This protocol may be used to study exosome-based communication in
other experimental settings as well. In addition, Inbar Azoulay-Alfaguter and Adam Mor
provideprotocolsfor theisolationandcharacterizationofTlymphocyte-derivedexosomes
using mass spectrometry. Particularly, they describe a centrifugation approach, combined
with mass spectrometry characterization, as a means to study exosomes derived from
primary human T lymphocytes. As mass spectrometry is a very sensitive method, this
protocolcanbeappliedwhenlimitedsamplesareavailable.
Three-dimensionalculturesarebetterabletoreflectthetumormicroenvironmentthan
two-dimensionalmonolayercultures, byfacilitating cell-cellinteractions in theappropriate
spatial dimensions. Tanya N. Augustine describes the isolation and co-culture of immune
cells with tumor cell lines in a three-dimensional system in a biologically relevant scaffold
facilitated by a basement membrane extract. This protocol allows for the assessment of
immune-tumor cell interactions in spatial dimensions that reflect the in vivo tumor micro-
environment. This protocol may be adapted for different cell types, and for determining a
responsetotherapeuticagents.
Continuing on the theme of intercellular communication, Monk et al. describe meth-
odologiesfortheco-cultureofmatureadipocytes(differentiated3T3-L1pre-adipocytecell
line)withprimaryimmunecellsubsetspurifiedfrommousesplenicmononuclearcellsusing
magneticMicroBeadpositiveselection.MicroBead-basedpositiveselectionmaybeusedto
purify multiple immune cell populations sequentially from a single mouse spleen, thereby
providing diversity in the types of immune cells that can be co-cultured with adipocytes.
Additionally, the authors provide the experimental procedures for co-culturing adipocytes
and immune cells in two different co-culture systems, including a cell contact-dependent
viii Preface
co-culture system wherein the cells are in direct physical contact, as well as a cell contact-
independent,solublemediator-drivenco-culturesystem,whereinatranswellsemipermeable
membranephysicallyseparatesthecells.
Ghia and colleagues have elaborated methods for the isolation of macrophages from a
varietyofmurinesources,includingperitoneal,bonemarrow-derived,and alveolar macro-
phages, which are extensively used to explore both the immunobiology and pathophysiol-
ogyofseveraldiseases.Inaddition,theauthorsdescribethephenotypiccharacterizationof
polarized human monocytic THP-1-derived macrophages and murine RAW264.7 cells
(a macrophage cell line). Ikeogu et al. describe methods for isolation and preparation of
bone marrow-derived immune cells for metabolic analysis, including macrophages, den-
driticcells,andneutrophilsfrommice.
The posttranslational modifications by ADP-ribosylation and phosphorylation are
important regulators of cellular pathways. While mass spectrometry-based methods for
the study of protein phosphorylation are well developed, protein ADP-ribosylation meth-
odologiesarestillindevelopment.Nita-Lazarandcolleaguesdescribeanimmobilizedmetal
affinity chromatography: a phosphoenrichment matrix-based method to enrich
ADP-ribosylated peptides, which have been cleaved down to their phosphoribose attach-
ment sites by a phosphodiesterase, thus isolating the ADP-ribosylated and phosphorylated
proteomes simultaneously for their quantitative analysis. Importantly, this protocol allows
the achievement of a robust and relative quantification of changes in the posttranslational
modification using dimethyl labeling, a straightforward and economical choice, which can
then be used on lysate from any cell type, including primary tissue. The protocol has been
optimizedtoworkinADP-ribosylation-compatiblebuffersandwithaprotease-ladenlysate
fromthemacrophagecells.
As mentioned above, cellular metabolism plays a central role in the activation and
effector functions of macrophages. Intracellular pathogens subvert the immune functions
ofmacrophagestoestablishaninfectionbymodulatingthemetabolismofthemacrophages.
Cumminget al.describehow theSeahorseExtracellular Flux analyzer(XF)can beused to
studychangesinthebioenergeticmetabolismofthemacrophagesinducedbyinfectionwith
mycobacteria. The XF simultaneously measures the oxygen consumption and extracellular
acidificationofthemacrophagesnoninvasivelyinrealtime,andtogether withtheaddition
of metabolic modulators, substrates, and inhibitors enables measurements of the rates of
oxidativephosphorylation,glycolysis,fattyacidoxidation,andATPproduction.
Anotherimportantimmunecelltypeisdendriticcells(DCs),whichserveasthebridge
between innate and adaptive immunity, and which are promising therapeutic targets for
cancer and immune-mediated disorders. This can be achieved by differentiating them into
eitherimmunogenicortolerogenicDCsbymodulatingtheirmetabolicpathways(including
glycolysis,oxidativephosphorylation,andfattyacidmetabolism)toorchestratetheirdesired
function. Thus, understanding the metabolic regulation of DC subsets and functions not
only will improve our understanding of DC biology and immune regulation, but can also
open up opportunities for treating immune-mediated ailments and cancer by adjusting
endogenous T-cell responses through DC-based immunotherapies. Wei et al. describe a
method to analyze this dichotomous metabolic reprogramming of DCs for generating a
reliable and effective DC cell therapy product. Particularly, by using a pharmacological
nuclear factor (Nrf2) activator as an example, they illustrate the metabolic profile of
tolerogenicDCs.
Preface ix
The mitochondrial membrane potential (Δψ) is an established indicator of the func-
tional metabolic status of mitochondria, which accounts for approximately 90% of all
available ATP for cellular activities. There are several experimental approaches to measure
Δψ levels,ranging fromfluorometricevaluationstoelectrochemicalprobes.Teodoroetal.
describetheevaluationofthemitochondrialmembranepotentialusingfluorescentdyesora
membrane-permeable cation (TPP+) electrode in isolated mitochondria and intact cells.
Moreover,theauthorsdiscusstheadvantagesanddisadvantagesofseveralofthesemethods,
ranging from one method that is dependent on the movement of a particular ion, tetra-
phenylphosphonium(TPP+)withaselectiveelectrode,totheselectionofafluorescentdye
from various types to achieve the same goal. These methods are highly sensitive, fast,
accurate, and a simple mode of evaluation of Δψ levels in respiring mitochondria, either
isolatedorstillinsidethecell.
Apartfromcellularmetabolism,mitochondrialdynamics(i.e.,mitochondrialfissionand
fusion) coincide with effectors and memory T-cell differentiation, resulting in metabolic
reprogramming. In general, freshly collected immune cells are preferred for such measure-
ments,asfrozencellsarenotconsideredidealforimmunometabolicanalyses.However,the
useoffreshlycollectedclinicalsamplesisnotalwayspossibleduetothelogisticdifficultiesof
having to complete analyses within a few hours of blood collection. Clovis Palmer and
coworkersdescribemethodsfor themultiparametric analysis ofmitochondrialdynamicsin
Tcells from cryopreserved peripheral blood mononuclear cells. They have optimized and
validatedasimplecryopreservationprotocolforperipheralbloodmononuclearcells,yield-
ing an astonishing >95% cellular viability, and preserved metabolic and immunologic
properties.Bycombiningfluorescentdyeswithcellsurfaceantibodies,theauthorsdemon-
strate how to analyze mitochondrial density, membrane potential, and reactive oxygen
speciesproductioninCD4andCD8Tcellsfromcryopreservedclinicalsamples.
Finally,Xuetal.describemethodstomeasureglucosehomeostasisatthesystemiclevel
(which is an integral component of immunometabolism) by measuring blood glucose
disposal and insulin sensitivity utilizing glucose tolerance and insulin tolerance tests. The
authors also provide valuable tips for consideration while performing these tests, as well as
datapresentationandinterpretation.
In addition to the previously discussed protocol chapters, opinion and review chapters
havebeenincluded withinthis book,relating to fundamentalaspectsinthe field ofimmu-
nometabolismandtheirimplications.Thefirstchapteristitled“ImmunometabolismandIts
Potentialto ImproveCurrentLimitations ofImmunotherapy” and isauthoredbyAndrew
Sheppard and Joanne Lysaght. The authors have provided an excellent account of the
promising discoveries made in this field, and future directions in the field can move in to
enhancetherapeuticeffectiveness.ThesecondchapterisauthoredbyMishraetal.,inwhich
the authors have highlighted the need to advance our understanding of sex differences in
thisfield,hencethetitle“SexDifferencesinImmunometabolism:AnUnexploredArea.”It
is anticipated that these two thought-provoking opinion chapters, along with a variety of
experimental protocols, will provide a valuable source of information and motivation for
researchersinthisemergingandpromisingfieldofimmunometabolism.
Iamindebtedtoalloftheauthorsforspendingtheirvaluabletimetocontributetothis
book. Importantly, I would like to thank John Walker, the series editor of Methods in
Molecular Biology, for the opportunity, as well as his guidance and help during the whole
