New Insights Into the Immune System Using Dirty Mice Sara E

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New Insights into the Immune System Using Dirty Mice Sara E. Hamilton, Vladimir P. Badovinac, Lalit K. Beura, Mark Pierson, Stephen C. Jameson, David Masopust and This information is current as Thomas S. Griffith of September 27, 2021. J Immunol 2020; 205:3-11; ; doi: 10.4049/jimmunol.2000171 http://www.jimmunol.org/content/205/1/3 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. New Insights into the Immune System Using Dirty Mice ,†,‡,x {,‖,# Sara E. Hamilton,* Vladimirx P. Badovinac,x Lalit K. Beura,** Mark Pierson,*x xx Stephen C. Jameson,*,†,‡, David Masopust,†,‡, ,†† and Thomas S. Griffith†,‡, ,‡‡, The mouse (Mus musculus) is the dominant organism then is how to improve the value of experimental rodent re- used to investigate the mechanisms behind complex search to increase the success rate of translating preclinical immunological responses because of their genetic sim- findings to the clinic. One option is for experimental im- ilarity to humans and our ability to manipulate those munologists to supplement existing or developing new models genetics to understand downstream function. Indeed, with variables known or expected to influence the readout, our knowledge of immune system development, re- outcome, and/or conclusions. Unfortunately, it is also fair to sponse to infection, and ways to therapeutically manip- note that most of the envisioned improvements will likely increase the complexity, cost, and, in some cases, limit the ulate the immune response to combat disease were, in Downloaded from large part, delineated in the mouse. Despite the power number of researchers able to fulfill the new model require- of mouse-based immunology research, the translational ments. In this review, we will discuss new insights into im- efficacy of many new therapies from mouse to human munology research using models based on mice with a history is far from ideal. Recent data have highlighted how the of microbial exposure. Humans are exposed to pathogenic and commensal microbes naive, neonate-like immune system of specific pathogen–

on a daily basis from birth, which (together with vaccination) http://www.jimmunol.org/ free mice differs dramatically in composition and mature the immune system to provide long-lasting protection function to mice living under barrier-free conditions against future microbial challenge. Immunological memory is (i.e., “dirty” mice). In this review, we discuss major minimal in most mice used in biomedical research because of findings to date and challenges faced when using specific pathogen–free (SPF) housing conditions in contrast to dirty mice and specific areas of immunology research the experienced immune systems of wild mice or mice obtained that may benefit from using animals with robust and from neighborhood pet stores [hereafter collectively referred to varied microbial exposure. The Journal of Immu- as “dirty” mice (4)]. The primary (and most obvious) feature of nology, 2020, 205: 3–11. dirty mice that distinguishes them from traditional SPF laboratory mice is that their exposure to multiple mouse pathogens by guest on September 27, 2021 aboratory mouse models represent an undeniable (which are normally excluded under SPF conditions) was asset in our understanding of immunology and hu- found to mature the murine immune system to more closely L man disease. Mice and humans share .90% ge- resemble that seen in adult humans (4–6). It is now appreciated netic homology (1), and their usage has led to the discovery that the baseline status of the immune system can have sig- of a significant number of genes and pathways fundamental nificant implications on the cellular composition and intensity for understanding disease processes and designing effective of subsequent immune responses. Our group published data treatment. Additionally, the power to transfer immune cells outlining the use of dirty mice to evaluate the composition and between hosts, ability to create genetic knockout strains, basic function of the immune system to challenge (compared conditional and inducible deletions, and transgenics are all with SPF mice), but additional work from a number of labo- important advantages to study disease processes within a fairly ratories has reinforced the concept and value of using dirty short timeline. However, many examples now exist whereby mice in preclinical investigation [Table I (4–10)]. Interest- use of the laboratory mouse has misdirected clinical ap- ingly, each publication has used slight variations in method- proaches, failing to positively impact humans and costing ology to generate the dirty mice used for study. The following millions of research dollars in the process (2, 3). The question information will be based on our experience with and data

*Department of Laboratory Medicine and Pathology, University of Minnesota, Minne- This work was supported by National Institute of Allergy and Infectious Diseases, apolis, MN 55455; †Microbiology, Immunology, and Cancer Biology Ph.D. Program, National Institutes of Health (NIH) Grants AI116678 (to S.E.H.), AI147064 (to University of Minnesota, Minneapolis, MN 55455; ‡Center for Immunology, Univer- V.P.B.), and AI084913 (to D.M.); National Institute of General Medical Sciences, x sity of Minnesota, Minneapolis, MN 55455; Masonic Cancer Center, University of NIH Grants GM134880 (to V.P.B.) and GM115462 (to T.S.G.); and U.S. Department { Minnesota, Minneapolis, MN 55455; Department of Pathology, University of Iowa, of Veterans Affairs Merit Review Award I01BX001324 (to T.S.G.). ‖ Iowa City, IA 52242; Department of Microbiology and Immunology, University of Address correspondence and reprint requests to Dr. Thomas S. Griffith, Department of Iowa, Iowa City, IA 52242; #Interdisciplinary Graduate Program in Immunology, Uni- Urology, University of Minnesota, MMC 394 Mayo, 420 Delaware Street SE, Minne- versity of Iowa, Iowa City, IA 52242; **Department of Molecular Microbiology and †† apolis, MN 55455. E-mail address: tgriffi[email protected] Immunology, Brown University, Providence, RI 02912; Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455; ‡‡Department of Abbreviations used in this article: CLP, cecal ligation and puncture; SPF, specific xx Urology, University of Minnesota, Minneapolis, MN 55455; and Minneapolis Vet- pathogen–free. erans Affairs Health Care System, Minneapolis, MN 55417 Ó ORCIDs: 0000-0002-3768-7622 (S.E.H.); 0000-0003-3180-2439 (V.P.B.); 0000- Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 0001-9137-1146 (S.C.J.); 0000-0002-7205-9859 (T.S.G.). Received for publication February 13, 2020. Accepted for publication May 4, 2020. www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000171 4 BRIEF REVIEWS: IMPACT AND FUTURE USE OF DIRTY MICE IN RESEARCH

Table I. Mouse models of microbial exposure

Model Strengths Weaknesses Human Genetic diversity (outbred) Significant regulations limit in vivo hypothesis-testing studies Experienced immune system Males and females can be used to address sex as a biological variable SPF mouse Can be inbred or outbred Naive immune system Infection history is controllable Microbiome is more uniform Numerous strains readily available to identify and interrogate Can be highly susceptible to pathogens immune cells Male and female mice can be used to address sex as a biological variable Wild mouse Natural pathogen exposure since birth Unknown infection history Diverse microbiome Unknown genetics and age Experienced immune system leads to enhanced protection to Increased cost for dedicated housing new infections Mice must be caught and may carry pathogens dangerous to humans (e.g., hantavirus) Cohoused with pet Can be inbred or outbred Increased cost for dedicated housing (e.g., BSL-3) store mice Can be age-matched with SPF mice Microbial exposure can vary with each cohort Downloaded from Natural pathogen exposure Cohousing can result in death depending on the pet store mouse used Conversion of the naive mouse immune system to a mature/ Unknown variation in microbiome composition effector phenotype is efficient (,10% of cohoused mice experience a poor conversion) and robust Restricted to female mice Sequential infection Infection history is known Requires prior selection of a “correct” set of pathogens http://www.jimmunol.org/ Using known pathogens draws on a larger pool of knowledge Uses laboratory defined routes of exposure instead of natural to interpret results routes Can be done with BSL1- and BSL2-level pathogens Milder conversion than observed with other methods Male and female mice can be used Exposure to dirty Mice rarely die or develop conditions that require euthanasia Fomite-generating pet store mice lose their ability to convert bedding (as seen in cohoused model) SPF mice over time, which requires additional pet store mice (fomites) and can create a higher percentage of low converted SPF mice Reduced variability: fomites allow for a more consistent and The intensity of conversion as measured is more erratic, and the uniform exposure over different cages with a reliable transfer variety of pathogens transferred is different from with of pathogens cohousing

Male and female mice can be used by guest on September 27, 2021 Natural microbiota Mice live in a more natural outdoor environment In rewilding model, outdoor enclosures must be constructed transfer/ that prevent escape and deter natural predators rewilding Phenotype is transferred from one generation to the next, In wildling model, wild mice must be captured, and embryo aiding in standardization transfers are technically challenging Natural pathogen exposure since birth Unknown variation in microbiome composition Male and female offspring mice can be used Increased cost for dedicated housing The models highlighted in the text are compared with traditional SPF mice and “dirty” humans. generated using our pet store mouse cohousing model, but we assessment primarily detects Ab generated after exposure to a will highlight various aspects of the other dirty mouse models set of well-defined viruses (e.g., parvovirus, mouse hepatitis used throughout our discussion. virus, and murine CMV) and a small number of bacteria, parasites, and fungi. Hence, it is also likely the pet store mice Models of microbially experienced dirty mice harbor a number of microbes that are not detected by the Cohousing model. To separate genetic and environmental effects most commonly used serological tests. on the cellular composition and function of the immune During the first week of cohousing, there is rapid expansion system, we have relied heavily on a model in which age-matched of the T cell compartment along with transition from a pre- cohorts of inbred strains of (initially SPF) laboratory mice are dominantly naive (CD44loCD62Lhi) to an activated/memory maintained in SPF housing conditions or housed in the same phenotype (CD44hiCD62Llo). The laboratory mice lose ∼10% cage with a commercial pet store mouse raised without of their body weight during the initial 14 d of cohousing, and barrier housing. This system is similar to the one used by we see ∼20% mortality rate (mostly within the first 21 d of Lindner et al. (11), who cohoused SPF mice with wild- cohousing; S.E. Hamilton, L.K. Beura, M. Pierson, unpub- caught mice to study how symbiotic host–microbe interactions lished data; and Ref. 4). By 6 wk, the number of T cells sta- within the gut modulate secretory Ab production. Regardless of bilize, and the mice do not appear overtly ill. After 60 d of thetypeofdirtymouseused,thecohousingmodelpermitsthe cohousing, 30–80% of the T cells in the blood have an direct comparison of the immune systems in the SPF and activated/memory phenotype (CD44hiCD62Llo) as opposed to microbially experienced, genetically identical, age-matched 10–15% in SPF mice (4, 12). In addition, the frequency of mice. Variation exists in the variety of microbes the pet KLRG1-expressing cells, which exist at only ∼1% in SPF mice, storemicecarry,andtheextentofmicrobialexposurecan, increases to 20–40% of the CD8 T cell compartment in most in part, be measured by serological assays and microbiome mice after cohousing (4, 12). These features have been fairly sequencing (4, 12). It is important to note that serological reproducible in multiple strains of inbred (i.e., C57BL/6, The Journal of Immunology 5

BALB/c, and C3H) and outbred Swiss Webster cohorts of of laboratory mice is significantly different from wild- mice (S.E. Hamilton, L.K. Beura, M. Pierson, and T.S. caught mice (11). Recent work from Rosshart and Rehermann Griffith, unpublished observations). Additional immuno- (10) has revealed just how much the wild microbiome can logical changes with cohousing include increased numbers of affect host immune fitness. Transfer of the natural commensal immune cells within nonlymphoid tissues (4), increased IgA microbes from wild mice to laboratory mice, either by repertoire diversity (11), increased circulating inflammatory gavage or naturally from mother to offspring, created a cytokines and chemokines (12), and increased numbers of matured host circulating immune system that conferred greater neutrophil and macrophage populations in the blood [all resistance to infection. Interestingly, C57BL/6 mice born compared with SPF mice (12)]. Importantly, we have not from pseudopregnant wild female mice (also known as observed increased expression of markers that indicate ex- “wildling” mice) replicated human immune responses when haustion (e.g., PD-1 or LAG3) on the T cells in cohoused used in preclinical models that failed clinically. The need for mice, suggesting effector function is likely maintained. capturing wild mice and performing embryo transfers into Sequential infection model. A more regulated and defined way to pseudopregnant females can make this model somewhat generate mice with an experienced immune system involves the more complex than others, but the fact that the natural sequential infection of SPF mice with a panel of known ex- microbiota are stable over numerous generations suggests perimental pathogens, such as the model used by Reese et al. (9) commensal transfer from wild mother to wildling offspring in which laboratory mice were infected with mouse hepatitis is sufficient to obtain a phenotypically and functionally more virus, murine CMV, influenza, and Heligmosomoides polygyrus. mature immune system. In a related model, Graham and Downloaded from In contrast to the bolus and likely asynchronous exposure to colleagues (24) simply transferred laboratory mice to an microbes experienced during cohousing with pet store mice, outdoor enclosure that exposed the mice to soil, vegetation, the controlled sequential infection to a panel of chosen and weather in a more natural habitat (25, 26). These microbes (using experimentally defined doses and routes “rewilded” mice showed maturation/differentiation of the of infection) greatly improves consistency within and between T cell compartment, increases in circulating granulocytes, experiments. Furthermore, the use of known pathogens allows and alterations in gut microbiota similar to what has been http://www.jimmunol.org/ the researcher to draw upon a larger pool of reagents and observed in other dirty mouse models. Interestingly, the previously published data specific to each pathogen used to increase in granulocytes related to increased gut colonization investigate immune system function and interpret results. by several fungal taxa (most notably Aspergillus species) (26). Sequential infection models using well-established experimental It is unclear if the gut microbiota changes that occur BSL1 and BSL2 pathogens also eliminate the need for special during rewilding will persist between generations, as seen in housing facilities (i.e., BSL3 for pet store mice) and procedures the wildling model. Moreover, construction of the outdoor to prevent the spread of unknown pathogens. Unfortunately, facility may be beyond the means for some. Regardless, the

there is not enough data on hand to know the extent to which compelling data generated using the wildling or rewilding by guest on September 27, 2021 infection with a defined, limited set of pathogens (in the models further demonstrate the increased similarity between absence of the commensal microbe transfer that occurs with microbially experienced mice with humans and the power of cohousing) fully or adequately replicates the immunological using them in “transitional–translational” studies. experience of humans. Furthermore, it is difficult to determine what combinations of laboratory-strain pathogens would be Challenges in using dirty mice appropriate and adequate to achieve the same impact as what Housing. Despite the physiological benefits of using dirty mice occurs with a more diverse and natural microbial exposure to study the immune system, there are several challenges to history. These caveats being raised, it is still noteworthy that using a mouse model with varied microbial experience. The the gene expression profiles of sequentially infected mice do first is logistical in that most facilities that house mice for more closely match humans than SPF mice, and many of the research work diligently to exclude select pathogens (e.g., phenotypic changes to immune cells noted in the cohousing mouse hepatitis virus, Mycoplasma pulmonis, Clostridium model were also observed (5). piliforme, and Enchephalotozoon cuniculi) normally present Natural microbiota transfer model. Human beings, like other in the wild. The desire to restrict/eliminate the microbial mammals, are colonized by microbes at all epithelial barriers contaminants commonly found in many institutional from birth (13). Being the largest body surface exposed to the animal facilities was initiated in the 1960s to increase the environmental factors, the gastrointestinal tract is populated reproducibility of the data generated (e.g., within groups with a myriad of microorganisms (mostly bacteria, fungi, and in a single experiment, experiment to experiment, and viruses) that vary along the gastrointestinal tract because institution to institution) and limit the number of unknown of unique physiologic conditions (14–17). The composition experimental variables, and the call for improved cleanliness of the gut microbial communities can be altered in a variety of has only intensified over time (27). Ways in which to ways, including antibiotic treatment, diet, and comorbidities overcome this logistical hurdle include housing dirty mice associated with disease (18, 19). Work from the last 25 y has in barrier-free facilities at distant locations, creating new unveiled how alterations in the gut microbiome can have outdoor spaces (24), or increasing the containment of dirty profound effects on the function of the systemic immune mice by working at BSL3 level. The main reason for having system (20, 21). Moreover, advances in genomic sequencing such elaborate housing for the dirty mice as well as restrict the technology have given researchers the tools to precisely define traffic pattern of the investigator (i.e., enter SPF housing the different microbes within the gut. Genetically identical before interacting with the dirty colony on the same day) is laboratory mice from different vendors have unique gut to prevent the spread of any microbes to the normal barrier microbiota (22, 23). Similarly, the bacterial gut microbiome facility. These housing restrictions frequently create new 6 BRIEF REVIEWS: IMPACT AND FUTURE USE OF DIRTY MICE IN RESEARCH

Table II. Microbial experience determined by serology testing Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

Percentage of positive mice tested is indicated. EDIM, epizootic diarrhea of infant mice.

financial hurdles for researchers, necessitating additional internal advantages associated with using fomites to make dirty male or external support to subsidize the work. In addition, some mice. From our experience, SPF mice exposed to fomites institutions may not have or be less willing to work with the rarely lose weight, die spontaneously, or develop conditions researcher to create suitable space to establish a colony of this that require euthanasia (as we have seen with cohousing; kind. Alternatively, outdoor housing (24) eliminates the needs S.E. Hamilton, L.K. Beura, and M. Pierson, unpublished for a BSL3 facility, but sufficient space and enclosure data). Bedding from a single cage of four to five pet store specifications must be available to ensure animal health and mice can provide enough fomites to convert 10–12 small safety. or 6–8 large cages of mice. Fomites also allow for more Analysis of male mice. Sex as a biological variable is becoming consistent and uniform exposure (even over several different increasingly appreciated as a critical factor in both basic im- cages), with a reliable transfer of pathogens. Despite these munology research and preclinical studies. Certain areas of strengths, we have found the efficiency and effectiveness of research can easily justify using a single sex of mouse for ex- fomites is not as robust as with cohousing. Fomite-generating periments, such as the exclusive use of male or female mice for pet store mice lose their ability to effectively passage microbes prostate or ovarian cancer research, respectively. Most other over time, which necessitates additional pet store mice and cancers or infectious diseases strike both males and females, can risk a higher percentage of poorly generated dirty mice. driving the push to perform experiments in mice of both sexes The intensity of conversion (i.e., frequency of effector/ to better capture the impact of sex differences on physiology. memory T cells in the blood) is more erratic than with Although the incorporation of mice of both sexes can be easily cohousing (S.E. Hamilton, L.K. Beura, and M. Pierson, done using the sequential infection or microbiota transfer unpublished data), and the variety of pathogens transferred (wildling) models of dirty mice, one difficulty of using the is slightly more limited (Table II). An alternative approach to cohousing model is the incompatibility (i.e., fighting) between fomite exposure would use cohousing with breeding inbred laboratory and pet store male mice. To overcome this obstacle, mice to get dirty male and female offspring, which would get we have found that exposing laboratory male mice to the dirty exposed in utero and beyond to various commensal and bedding (“fomites”) from pet store mice achieves the main pathogenic microbes; however, we do not yet have a full goal of maturing the immune systems in male laboratory account of whether this approach would achieve the same mice through natural microbial exposure. There are several phenotypic and functional changes within the immune system The Journal of Immunology 7 as seen in adult cohoused mice or the multigenerational effects immune reactivity. Vaccines are frequently less efficacious in seen in the natural microbiota transfer (wildling) model (10). individuals from developing countries where the number of Lack of control. Perhaps the biggest challenge to using dirty mice previous microbial encounters is increased compared with is becoming comfortable with the uncontrolled nature of people in developed areas of the world (33–35). The reasons natural microbial exposure. In addition to the variation in behind the depressed vaccine-induced response are not fully pathogenic and commensal microbes present in wild and pet understood and (as expected) difficult to tease apart in humans. store mice, not all microbes are efficiently transmitted to cage Despite the accepted influence of previous and ongoing partners, whereas others are more frequently transferred. This microbial exposures on the immune system, the SPF mouse variability creates a mixture of exposure for each individual continues to be a common initial model used in vaccine de- mouse, even within the same cage. Moreover, we are unable to velopment. Some investigators have attempted to deal with the identify every microbe the laboratory mouse has encountered issue of an ongoing infection by challenging SPF mice with a during cohousing with pet store mice using the most robust single pathogen (typically establishing a chronic viral or par- assays currently available, and seroconversion is dependent on a asite infection), followed by the initiation of a new immune number of factors (e.g., dose, pathogen, and age and genetic response (30, 36–39). These studies have revealed important composition of the infected animal). Some may view these changes in both innate and adaptive responses dealt by on- unknowns as an affront to our scientific instinct to control going infection but are limited by the singularity of the ex- variables and attempt to discredit the validity of the model, but posure. The use of dirty inbred (and potentially outbred) mice it can simultaneously be argued that this variability is a more may reveal additional, hitherto unappreciated, differences in Downloaded from realistic representation of the differential exposure humans the response to vaccines, perhaps highlighting pathways to encounter by living in the natural environment. Despite this improved adjuvants or methods of vaccination that are more variability, we do not find excessively large numbers of mice per likely to be successful. Because dirty mice harbor vastly in- experimental group (five to ten cohoused versus three to five creased numbers of resident memory T cells (similar to hu- SPF mice) are needed to achieve statistical significance. mans) (4), they may represent a more relevant animal model

for the testing of vaccines aimed at establishing memory cells http://www.jimmunol.org/ Research areas that beneﬁt from the use of dirty mice in barrier tissues, which is likely to be critical for diseases like Data generated to date suggest the immune system of dirty HIV, tuberculosis, and inﬂuenza. mice is a more accurate model of the adult human immune In the realm of vaccine development/testing, the use of mice system (4, 6, 9–12, 24). Consequently, dirty mice are now offers researchers numerous advantages. For basic investiga- recognized as an important tool for improving the expediency tion, vaccine testing in mice eliminates the expense and time of translational research. It is important to emphasize, how- constraints needed for the GMP manufacture, Food and Drug ever, that much work still needs to be done to rigorously show Administration approval, and monitoring that would be

the translational applicability of the dirty mouse model, as needed for human application. Interrogation of the murine by guest on September 27, 2021 well as its breadth within immunology. One immediate ex- immune system during vaccine testing can be done at great tension of future dirty mouse models will include settings in depth, in which it is possible to perform high-throughput which work with inbred strains of mice will be supplemented analysis of multiple time points and tissues that may con- with outbred cohorts (or Collaborative Cross) mice to more tain the critical immune cell populations involved in the accurately reflect the variations in immune outcomes in the protective response. Moreover, studies that rely on gene ma- genetically diverse human population. However, the cohous- nipulation (e.g., use of knockout or reporter mice) give the ing approach may not feasible given that the Collaborative researcher the opportunity to mechanistically dissect the Cross mice consist of a large number of genetically different vaccine response in mice. Challenge studies designed to test mice. Rather, SPF Collaborative Cross lines should be initially vaccine effectiveness are much more feasible in the mouse, screened to define a particular phenotype/function/gene locus although it is possible to conduct such investigation in hu- of interest (28) before moving a specific Collaborative Cross mans. Despite these advantages, vaccine testing in humans mouse line into a dirty environment. The following sections must still be done, and the advanced technologies available in highlight areas of research that seem particularly pertinent to the current “-omics” era are making human vaccine testing test how the features of dirty mice echo observations in hu- more feasible. There are human versus mouse species differ- mans. We will also consider how this model could best be ences that have nothing to do with microbial experience, such leveraged to improve vaccines, medications, and treatments as species-specific tropisms, that can affect vaccine potency for diseases and conditions that have not been adequately and help to justify human vaccine testing. The addition of revealed through research in SPF mice. dirty mice to the vaccine toolbox will hopefully advance the Vaccine efficacy. Infectious history clearly impacts subsequent testing possibilities prior to human evaluation. immune responses even to discordant infections. Twin studies Trained immunity. Host immunity has been classically divided show immune system variability is dictated in large part by into two arms: the quick-acting, Ag-independent innate acquired, and not only genetic, factors (29). Furthermore, response and the slower, but Ag specific, adaptive response. chronic viral infections are prevalent in humans, which stim- Moreover, it was long thought that only adaptive immunity ulate ongoing immune responses (30). Aside from Ag-specific wasabletoestablishmemory.Datafromavarietyof immunity, increased numbers of Ag-experienced immune cells organisms, ranging from plants to invertebrates to mice can improve Ag-independent alarm responses, which are a and humans, now clearly demonstrate, however, that prior potent mechanism of early pathogen detection and amplification microbial exposure can also train innate immune cells of immune responses to control infection (31, 32). However, (e.g., neutrophils, monocytes/macrophages, and NK cells) to extensive immune experience does not always predict improved exhibit memory and mediate resistance to secondary challenges 8 BRIEF REVIEWS: IMPACT AND FUTURE USE OF DIRTY MICE IN RESEARCH

(40, 41). Dirty mice exhibit increases in neutrophils, monocytes/ severe weight loss and increased acute mortality after sepsis macrophages, and NK cells, as well as increased expression on induction. The increased morbidity/mortality in cohoused TLR on these populations (10, 12). It remains to be mice after CLP correlated with exaggerated acute hyper- determined whether these increases are driven by pathogenic inflammation, as seen by increased serum IL-1b,IL-6, infection or commensal colonization (or both), but they IFN-g, and TNF post-CLP. IL-6 levels in septic humans suggest these populations have the capacity to respond more correlate with disease severity and outcomes (75, 76), vigorously to subsequent challenges. To complement the whereas TNF and IL-1b are primary mediators of current models using bacillus Calmette–Gue´rin or b-glucan inflammation-induced activation of coagulation (77) dur- (42), it would be intriguing to use dirty mice to investigate ing sepsis. Administration of ceftriaxone (50 mg/kg) and some of the underinvestigated areas in trained immunity metronidazole (30 mg/kg) (78) at 12 and 24 h after CLP research. For example, dirty mice could be used to better did not improve survival of cohoused mice, suggesting that define the role of the adaptive immune system in the once the hyperinflammatory response is triggered the pres- generation and maintenance of memory within innate enceoftheinducingmicrobesbecomeslessimportant. immune cells because of the significant increase in mature, Similar outcomes were observed when we used a cecal Ag-experienced T cells in dirty mice. Furthermore, dirty mice slurry or LPS endotoxemia model of sepsis. As part of the can be used to help delineate the temporal, cellular, metabolic, data published recently by Rosshart et al. (10) the thera- and epigenetic dynamics of innate memory responses. peutic benefit of anti-TNF treatment during septic shock

Sepsis. Sepsis is a significant public health burden, striking over was tested in SPF laboratory and dirty wildling mice. Downloaded from 1 million Americans annually (43–45). Sepsis was recently Prophylactic administration of anti-TNF mAb was able to redefined as a “life-threatening organ dysfunction caused by protect SPF laboratory mice from lethal LPS endotoxemia, a dysregulated host response to infection” (46). Early stages but this therapy failed to rescue the wildlings. These data of sepsis are marked by hyperinflammation driven by were consistent with clinical results, demonstrating the proinflammatory cytokines (i.e., IL-1b,IL-6,IFN-g,and inability of anti-TNF therapy to improve survival in septic TNF) (47–49). Mouse models of sepsis have been used to shock patients (79). However, it is important to note the http://www.jimmunol.org/ define and understand the pathophysiological complications anti-TNF mAb was administered to the mice prior to LPS that develop during a septic event (50, 51), as well as challenge, whereas patients were given anti-TNF therapy developing therapeutic modalities to counteract the profound after sepsis onset. Thus, although the data with the LPS- immunological changes that contribute to sepsis-induced treated wildling mice suggest they phenocopy sepsis patients mortality (47, 52, 53). We have been interested in studying in regard to their lack of response to TNF neutralization, a variety of immunological parameters altered in the septic these data highlight the potential power of using microbially host (54–63) and (like most sepsis researchers) have relied on experienced mice as a transitional step between traditional

SPF mice for these experiments. Unfortunately, very few of SPF mice and humans in the evaluation of immunomodu- by guest on September 27, 2021 the pharmacological therapies demonstrating efficacy in latory therapeutics designed to lessen the cytokine storm and preclinical sepsis models have shown similar success in immune suppression induced by sepsis, with the intention of humans, resulting in significant scrutiny of mouse models improving patient outcomes. of sepsis. Although SPF housing of laboratory mice has Future research directions been instrumental in increasing experimental reproducibility in the investigation of sepsis-driven immunoparalysis, it has It is likely that additional areas of immunology research will simultaneously further distanced the mouse as a model from benefit from using dirty mice in the future. Although the list of humans largely because SPF mice live their lives with limited future research directions with dirty mice is unlimited, the exposure to mouse pathogens (64). There have been a handful following areas are just a few of the many in which preliminary of reports using humanized NOD SCID gC mice to evaluate pieces of data are on hand that demonstrate how the microbial the acute changes in leukocytes and circulating cytokines/ experience history of the host can potentially impact the way chemokines after sepsis induction (65–70), but species the immune system responds. incompatibility between some cytokines and their receptors, Tumorogenesis and immunotherapy. In the last 10–15 y, tumor absence of HLA molecules on host APCs, and potential for immunology has experienced an unprecedented explosion in low levels of hematopoietic cell engraftment, human immune research and the development of improved treatments. Im- cell development, and function have limited the ability of mune checkpoint therapy has revolutionized the way cancer humanized mice to faithfully replicate keys aspects of the can be treated, and it is clear these current (and future) means human immune system (71–73). of treating cancer have strong foundations in preclinical Data obtained from cohoused mice after systemic infection mouse modeling. Despite these successes, many current with virulent Listeria monocytogenes or malaria suggest mouse models are still heavily scrutinized because they can be microbially experienced hosts can more efficiently control poor at modeling tumor control, the impact of immuno- subsequent infections compared with SPF mice (4). With therapy, and the adverse events related to therapy that can this increased resistance to systemic experimental infection, result in humans. The use of humanized immunodeficient we used the cecal ligation and puncture (CLP) model of mice bearing human tumors (either patient-derived xenografts sepsis (74) to study the acute immune response to a sys- or human tumor cell lines) has given cancer researchers temic polymicrobial infection by gut commensals, expect- another model for examining the therapeutic benefit of ingcohousedmicetobemoreresistantthanSPFmice. various drugs under development, but these models have Even though the SPF and cohoused mice had equivalent their limitations, including the lack of function of some bacteremia after CLP, the cohoused mice exhibited more mouse cytokines on human immune cell development and The Journal of Immunology 9 function, the possibility of xenogeneic graft-versus-host Whether there are conserved pathways between the aging disease, and presence of host innate immunity. Recent immune system and the experienced one or these are two data have also highlighted the potential of harnessing the completely independent processes is an important line of immunological power in pathogen-specific tissue resident investigation to be considered moving forward. memory T cells to recognize and eliminate tumors (80–86). Neurodegenerative diseases. Over the last decade, it has become Inclusion of dirty mice in the preclinical repertoire of increasingly appreciated that most neurodegenerative diseases, reagents used to study tumor formation and/or de novo including Alzheimer disease, amyotrophic lateral sclerosis, and or therapy-driven immune responses to tumors may give Parkinson disease, stem from inflammatory processes in the researchers new insight into the dynamic tumor/immune CNS and the host immune system is intimately involved in the system relationship seen in adult humans with experienced disease pathogenesis (99). Various CNS-resident and -recruited immune systems (87, 88). The addition of dirty mice into immune cells directly participate in these neuroinflammatory tumor immunology may be especially useful with agents events. Although the cause for these highly complex pathologic designed to stimulate adaptive immune responses, as processes are likely multifactorial, there are several reports of T cell differentiation or the composition and/or magnitude evidence of viral and bacterial involvement (100–102). However, ofthecytokineresponsemaybequitedifferentfromthat most preclinical animal model studies of neurodegenerative seen in traditional SPF laboratory mice. diseases continue to use transgenic mice reared in the ultra- The hygiene hypothesis and responses to allergens. Considerable hygienic SPF conditions. Although these studies have provided attention has been given to the concept that the increased important information regarding basic aspects of disease process Downloaded from incidence of allergen sensitivity (including allergic asthma) in and changes in the immune system, therapies designed to industrialized nations reflects more limited exposure to mitigate immunopathogenesis have yet to translate to humans pathogens as a consequence of improved public health stan- even though they were efficacious in SPF mouse-based dards and efficient vaccination programs. Some epidemio- preclinical studies. Inclusion of more microbially enriched logical and experimental evidence supports this “hygiene dirty environments for rearing various transgenic mouse hypothesis” (89, 90), but it is still unclear to what extent the models has the potential to better recapitulate the disease http://www.jimmunol.org/ low incidence of allergic diseases in some populations reflects process as well as successful therapy design. the impact of diverse microbial experience versus exposure to a specific set of microbial factors. Most preclinical studies in Conclusions mice investigate how individual microbes or microbial prod- The use of dirty mice has advanced our understanding of the ucts affect the response to allergens in SPF mice, making it immune system and moved us closer to replicating human hard to extrapolate whether natural exposure to a diverse immune experience. Many more studies will be needed, in population of pathogens (as experienced by nearly all humans multiple models of infection, cancer, and other immune

and modeled in dirty mice) does or does not affect the inci- perturbations to comprehensively learn the unique features of by guest on September 27, 2021 dence or severity of allergic/asthmatic responses. In addition, these mice. Incorporating outbred or Collaborative Cross dirty it remains to be determined whether responses to allergens in mice in the future will also reflect variation of immune out- these populations are simply inhibited or, instead, are diverted comes in diverse populations and potentially uncover un- into distinct forms of immune reactivity that do not prompt derlying genetic causes. Critically, more studies are needed typical allergic/asthmatic diseases (91, 92). It will be interesting directly comparing data from humans given vaccines or to see to what extent and how the broad microbial exposure therapies alongside dirty and traditionally used SPF mice to experienced by dirty mice alters the immunological detection of determine if the dirty mouse model will consistently lead us to and response to allergens. more accurate conclusions about human immune responses. Aging. Normal aging results in both intrinsic changes to the immune system (e.g., thymic involution and loss of naive T cell Disclosures production) as well as accumulating exposures to vaccines and The authors have no financial conflicts of interest. microbes. These amassed changes frequently result in immune disorders related to either immunosenescence (an insufficient immune response) or inflammaging (an exacerbated immune References 1. Mural, R. J., M. D. Adams, E. W. Myers, H. O. Smith, G. L. Miklos, R. Wides, response) (93). Certain populations, such as HIV-infected A. Halpern, P. W. Li, G. G. Sutton, J. Nadeau, et al. 2002. A comparison of people, show early onset of age-related chronic disease and whole-genome shotgun-derived mouse chromosome 16 and the human genome. Science 296: 1661–1671. geriatric symptoms that appear unrelated to a specific microbial 2. Mak, I. W., N. Evaniew, and M. Ghert. 2014. Lost in translation: animal models exposure, suggesting the effects of aging could be accelerated or and clinical trials in cancer treatment. Am. J. Transl. Res. 6: 114–118. 3. Wong, C. H., K. W. Siah, and A. W. Lo. 2019. Estimation of clinical trial success compounded with some chronic infections (94, 95). Several rates and related parameters. [Published erratum appears in 2019 Biostatistics 20: commonalities have been noted between dirty mice and aged 366.] Biostatistics 20: 273–286. 4. Beura, L. K., S. E. Hamilton, K. Bi, J. M. Schenkel, O. A. Odumade, K. A. Casey, SPF mice, such as persistent, systemic inflammation, elevated E. A. Thompson, K. A. Fraser, P. C. Rosato, A. Filali-Mouhim, et al. 2016. numbers of memory T and B cells, exacerbated septic Normalizing the environment recapitulates adult human immune traits in labo- responses, and upregulation of senescence markers like ratory mice. Nature 532: 512–516. 5. Japp, A. S., K. Hoffmann, S. Schlickeiser, R. Glauben, C. Nikolaou, H. T. Maecker, KLRG1 (96). The increased number of senescent cells J. Braun, N. Matzmohr, B. Sawitzki, B. Siegmund, et al. 2017. Wild immunology found in aged mouse models can experimentally lead to a assessed by multidimensional mass cytometry. Cytometry A 91: 85–95. 6. Rosshart, S. P., B. G. Vassallo, D. Angeletti, D. S. Hutchinson, A. P. Morgan, number of age-related pathologies (97). The recent development K. Takeda, H. D. Hickman, J. A. McCulloch, J. H. Badger, N. J. Ajami, et al. of senolytic drugs to eliminate senescent cells is an area of active 2017. Wild mouse gut microbiota promotes host fitness and improves disease resistance. Cell 171: 1015–1028.e13. research (98), and it would be useful to test these clinical 7. Huggins, M. A., S. C. Jameson, and S. E. Hamilton. 2019. Embracing microbial compounds in dirty mouse models as a test of efficacy. exposure in mouse research. J. Leukoc. Biol. 105: 73–79. 10 BRIEF REVIEWS: IMPACT AND FUTURE USE OF DIRTY MICE IN RESEARCH

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