Mastomys natalensis Has a Cellular Immune Response Profile Distinct from Laboratory Mice

The multimammate mouse (Mastomys natalensis; M. natalensis) has been identified as a major reservoir for multiple human pathogens including Lassa virus (LASV), Leishmania spp., Yersinia spp., and Borrelia spp. Although M. natalensis are related to well-characterized mouse and rat species commonly used in laboratory models, there is an absence of established assays and reagents to study the host immune responses of M. natalensis. As a result, there are major limitations to our understanding of immunopathology and mechanisms of immunological pathogen control in this increasingly important rodent species. In the current study, a large panel of commercially available rodent reagents were screened to identify their cross-reactivity with M. natalensis. Using these reagents, ex vivo assays were established and optimized to evaluate lymphocyte proliferation and cytokine production by M. natalensis lymphocytes. In contrast to C57BL/6J mice, lymphocytes from M. natalensis were relatively non-responsive to common stimuli such as phytohaemagglutinin P and lipopolysaccharide. However, they readily responded to concanavalin A stimulation as indicated by proliferation and cytokine production. In summary, we describe lymphoproliferative and cytokine assays demonstrating that the cellular immune responses in M. natalensis to commonly used mitogens differ from a laboratory-bred mouse strain.


Introduction
Mastomys natalensis, a member of the Muridae family [1], has high prevalence across sub-Saharan Africa [2][3][4]. M. natalensis frequently lives in close association with humans and has been identified as a host reservoir for several zoonotic pathogens, including LASV [5][6][7], Leishmania major (L. major) [8], Borrelia spp. [9][10][11], and Yersinia pestis [12]. In contrast to humans, infection of M. natalensis by many of these zoonotic pathogens appears to be asymptomatic. How the immune system plays a role in pathogen persistence and clearance in these animals is unknown. An improved understanding of mechanisms by which M. natalensis controls microbial infection and transmission may lead to the development of novel intervention strategies to reduce zoonotic transmission to humans.
Laboratory mouse and rat models have provided invaluable insight into the pathology and immunobiology of many different pathogens [13][14][15][16][17][18]. However, it is becoming increasingly appreciated that many aspects of microbial immunobiology may differ in these established rodent models from those of wild rodent species serving as pathogen reservoirs. Further, the applicability of commercially available reagents and well-established immunological techniques, including flow cytometry and in vitro T-cell assays, commonly used to study immune responses in laboratory mice and rats have not been established for the study of M. natalensis immunity.
In the present study, we first aimed to determine if conventional reagents used in laboratory rodent studies could be used to trigger M. natalensis cells. We screened commercially available antibodies for use with M. natalensis splenic lymphocytes. Using identified reagents, we optimized in vitro assays for T-cell proliferation and the detection of IFN-γ and TNF-α production. We show that in response to well-defined stimuli, the activation potential of M. natalensis splenic lymphocytes differs substantially from those observed in C57BL/6J mice.

Animals
In this study, we used M. natalensis from an in-house breeding colony originally established from rodents captured in Doneguebougou, Mali [40]. C57BL/6J mice were obtained from Jackson Laboratory. For all experiments, we used 5-7 week old animals with equal sex distribution. The number of animals used for each experiment is indicated in the legends of the figures. M. natalensis were free of ectromelia virus, mouse rotavirus, lymphocytic choriomeningitis virus, mouse adenovirus, Sendai virus, mouse hepatitis virus, minute mouse virus, mouse parvovirus, mouse polyoma virus, mouse norovirus, Theiler's murine encephalomyelitis virus, Mycoplasma pulmonis, pinworms, and ectoparasites according to dirty bedding serology and filter EDx PCR testing (IDEXX BioAnalytics, Columbia, MO, USA). C57BL/6J mice were free of the above pathogens according to vendor reports. Animal studies were approved by the Institutional Animal Care and Use Committee and were conducted in compliance with all institutional and national guidelines for use and handling of animals.

Reagents and Antibodies
A list of reagents and antibodies is provided in Table 1.

Tissue Preparation
Spleens were harvested and placed in RPMI supplemented with 10% FBS and 2% of penicillin/streptomycin (ThermoFisher, Carlsbad, CA, USA), L-glutamine (Sigma, St. Louis, MO, USA), and 0.5 mM of β-2-mercaptoethanol (Sigma, St. Louis, MO, USA) (cRPMI). Tissues were individually dissociated at room temperature (RT) in Miltenyi dissociator C tubes (Miltenyi Biotec, San Diego, CA, USA) ( Table 1). Following dissociation, spleen homogenates were passed through a cell strainer (Fisher Scientific, Pennsylvania, PA, USA) and centrifuged at 700× g for 5 min. Red blood cell were lysed using 1× RBC Lysis Buffer according to manufacturer's instructions (eBioscience™ ThermoFisher, Carlsbad, CA, USA). Remaining cells were washed and resuspended in cRPMI, and cell counts were performed by mixing 10 µL of sample with 10 µL of 0.4% trypan blue solution. The mixture was loaded onto a chamber slide and counted using a Countess cell counter (Bio-Rad, Hercules, CA, USA).

Extracellular Staining for Flow Cytometry
Non-specific binding was blocked using TruStain (Biolegend, San Diego, CA, USA) for 10 min. Splenocytes were stained with T cell surface markers with rat anti-CD3, CD8, and CD4 for 30 min at 4 • C ( Table 1). The samples were analyzed on a BD FACS Symphony instrument (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo v10.

Cytometric Bead Array (CBA)
Splenocytes (1 × 10 6 cells/well) were incubated with or without ConA, LPS, or PHA mitogens for 24 h in cRPMI as technical duplicate replicates. Supernatants were collected and stored at −20 • C until use. TNF-α and IFN-γ release into the supernatant was measured by CBA using anti-mouse TNF-α and anti-rat IFN-γ antibodies according to the manufacturer's instructions ( Table 2).

Software and Statistical Analysis
Flow cytometry data were analyzed using FlowJo software v10 (Tree Star). Statistical analyses were performed using the Prism software v8 (GraphPad, San Diego, CA, USA). Wilcoxon non-parametric test and one-way ANOVA were used. Variance was similar between the groups being compared.

Commercial Rat and Mouse Antibodies Cross-React with M. natalensis T Cell Receptors and Intracellular Cytokines
Commercial rat and mouse antibodies against T cell receptors (CD3, CD8, CD4) and effector molecules (TNF-α and IFN-γ) were evaluated for their cross-reactivity with M. natalensis splenocytes (Table 3). Spleens from M. natalensis were harvested and stained with T cell receptor antibodies from different clones and analyzed by flow cytometry. We found that M. natalensis CD3 and CD8b receptors were recognized by rat anti-CD3 clone CD3-12 and rat anti-CD8b clone 341, respectively. No CD4 antibodies tested in this study cross-reacted with M. natalensis splenocytes (Table 3). In addition, we could demonstrate that M. natalensis IFN-γ and TNF-α cytokines were recognized by rat anti-IFN-γ, clone DB-1, and mouse anti-TNF-α clone MP6-XT22, respectively (Table 3).

ConA Mitogen Efficiently Induced M. natalensis T Cell Proliferation In Vitro
ConA [41,42], PHA [43][44][45], and LPS [46,47] are the most commonly used mitogens targeting lymphocytes as they do not require antigen presentation to activate T cells and have been used to describe general immune responses, such as proliferation and cytokine production, in these cell populations. To optimize the in vitro assay for the induction of T cell proliferation and differentiation into mature effector cells, spleens from M. natalensis were harvested and stimulated with these three mitogens and T cell proliferation was measured at different time points (0, 3, 4, 5, and 6 days) post-stimulation. Recombinant mouse IL-2 was added to a subset of these samples to assess the impact of IL-2 signaling on activated cell survival, which by itself failed to stimulate T-cell proliferation. The CTV-based assay has been used to quantify T-cell proliferation in response to different mitogens. Unstimulated splenocytes were used as negative controls.
In agreement with previous studies, LPS, ConA, and PHA mitogens induced proliferation among C57BL/6J splenic lymphocytes [42,43,47] independently of IL-2 (Figure 1a-c). In contrast to C57BL/6J, M. natalensis splenic lymphocytes stimulated with LPS did not proliferate with or without IL-2 (Figure 1a). We observed that M. natalensis cells stimulated with PHA proliferate only in the presence of recombinant mouse IL-2 ( Figure 1b). Finally, ConA was sufficient to enhance a strong CD3 + -T cell proliferation in both C57BL/6J and M. natalensis, and this effect was independent of IL-2 ( Figure 1c). Thus, ConA triggered the most efficient proliferation of M. natalensis CD3 + T cells compared to PHA and LPS mitogens.

Comparative Secretion of Effector Molecules in M. natalensis in Response to Stimuli
To further examine the differential immune response profiles by M. natalensis splenic lymphocytes, we assessed the level of cytokines in cell supernatant or secreting cells following mitogenic stimulation. M. natalensis splenic lymphocytes were stimulated with LPS, PHA, or ConA for 24 h. C57BL/6J splenic lymphocytes were used as a positive control. As described above, the presence of IL-2 can have an impact on T cell proliferation ( Figure 1b); therefore, we also included recombinant mouse IL-2 to a subset of samples to determine its impact on cytokine production. TNF-α and IFN-γ were detectable in the supernatant of secreting splenic lymphocytes from C57BL/6J mice when stimulated with mitogens as determined by mouse specific ELISA, CBA, and ELISpot kits. No detectable TNF-α and IFN-γ in cell culture supernatants from M. natalensis splenic lymphocytes was observed when assessed by ELISA (data not shown). However, both cytokines were detected in supernatant utilizing CBA (Table 2). Further, TNF-α was readily detected by ELISpot (Table 2).
We used CBA specific to rat or mouse to measure cytokine responses to different stimuli in M. natalensis and C57BL/6J rodents, respectively. C57BL/6J splenic lymphocytes responded to all stimuli, LPS and ConA, as indicated by the production of both TNF-α and IFN-γ, and no differences were observed in IL-2 treated groups. Among PHA-stimulated C57BL/6J cells, the production of both cytokines significantly decreased (p < 0.01) in the presence of IL-2 ( Figure 2a). In contrast, IL-2 did not impact C57BL/6J T-cell proliferation (Figure 1a-c) and M. natalensis stimulated cells only produce TNF-α in response to PHA but not IFN-γ independent of the presence of IL-2 ( Figure 2b). However, LPS and ConA treatment significantly increased the secretion of TNF-α and IFN-γ within 24 h by M. natalensis splenic lymphocyte. Addition of IL-2 did not significantly change the amount of either cytokine produced under these conditions (Figure 2a,c). Taken together, LPS and ConA efficiently induce both IFN-γ and TNF-α secretion independently of IL-2 in both C57BL/6J and M. natalensis.

Comparative Expression of Intracellular Cytokines in M. natalensis in Response to Different Stimuli
Next, we assessed the expression of effector molecules by M. natalensis CD3 + T cells in response to mitogens stimuli. Intracellular IFN-γ and TNF-α were not detected among M. natalensis splenic lymphocytes stimulated only with PMA/Iono in the presence of BFA for 6 h. Therefore, M. natalensis splenocytes were stimulated with ConA, LPS, or PHA for 6 days followed by re-stimulation with PMA/Iono in the presence of BFA (Figure 3b). All mitogens induced the expression of both IFN-γ and TNF-α among positive control C57BL/6J CD3 + T cells (Figure 3b,d). All three mitogens also induced the expression of TNF-α by M. natalensis CD3 + T cells, but only ConA induced significant expression of both IFN-γ and TNF-α (Figure 3b,c). Notably, as observed in studies assessing the proliferation potential, IL-2 did not impact the expression of either IFN-γ or TNF-α in the M. natalensis CD3 + cell population. Therefore, only ConA efficiently induced the expression of IFN-γ and TNF-α by M. natalensis CD3 + T cells as detected by intracellular cytokine staining.

Discussion
M. natalensis is a host for multiple emerging and re-emerging human pathogens (i.e., LASV, Leishmania spp., Yersinia spp., and Borrelia spp.). It is unknown how these rodents survive infection with these pathogens to serve as vectors for transmission to humans. Understanding this paradigm may ultimately help the development of new therapeutic strategies. T cells play an important role in the defense against microorganisms, in part by secreting key mediators which enable eradication of the infecting agent. Th1 T cells are characterized by their property to produce IFN-γ, TNF-α, and IL-2. These cells play a central role in mediating adaptive immune responses to microbial agents [21,23,32,33,35,48,49], tumor [50], inflammation, and autoimmune diseases [51,52]. Therefore, development of assays that assess these responses in M. natalensis-derived cells would provide valuable insight into our understanding of ongoing immune responses in this reservoir host.
To study the T cell-mediated immunity in M. natalensis, we identified, optimized, and established immunological techniques used for laboratory mice and rats to trigger T cell activation. M. natalensis splenocytes were stimulated with the classical mitogens (LPS, PHA, or ConA) that do not rely on antigen specificity or presentation to trigger proliferation and production of cytokines by these cells [41][42][43][44][45][46][47]. We found that M. natalensis CD3 + , CD8 + T cell markers, and IFN-γ were largely recognized by antibodies directed against rat proteins whereas TNF-α was detected by antibodies targeting mouse cytokines (Table 3). Approaches for measuring cytokines/chemokines associated with cytotoxic T-lymphocyte function in response to mitogenic stimuli in cell supernatant or cytokine-secreting cells were also assessed. We found that only CBA, a bead-based immunoassay, was capable of measuring both M. natalensis IFN-γ and TNF-α in cell culture supernatants after 24 h of stimulation with mitogens ( Figure 2). CBA, ELISA, and ELISpot assays all use primary (capture) and secondary (detection) antibodies. However, there are differences among these methods, such as their sensitivity to detect low frequency of cytokine-secreting cells (ELISpot) or cytokines release into cell culture (CBA and ELISA) [53][54][55]. In addition, we have demonstrated that M. natalensis IFN-γ is only detectable by a rat-IFN-γ CBA kit and TNF-α by mouse-TNF-α ELISpot and CBA kits. These results suggest that paired antibodies used to detect IFN-γ or TNF-α may differ in those assays or that some antibodies do not function due to lack of binding. This is supported by a recent phylogenetic study demonstrating that the genome of Mastomys coucha aligns to 90.1% with mouse and 85.5% with rat, supporting an intermediate position of M. natalensis in the rodent taxonomy [56].
In this study, we have demonstrated significant differences in T cell proliferation and cytokine production between M. natalensis and C57BL/6J splenic lymphocytes in response to different stimuli. It should be noted, however, that M. natalensis animals from our colony were recently derived from wild caught animals [40] and thus may harbor microorganisms distinct from laboratory C57BL/6J mice derived from a clean and defined laboratory environment. Therefore, M. natalensis immune responses to experimental mitogen stimulation may be reduced due to continuing exposure to microbial stimuli [57,58].While all mitogens induced C57BL/6J splenic lymphocytes proliferation, independent of IL-2, LPS was not effective at triggering proliferation of M. natalensis splenic T cells regardless of whether exogenous IL-2 was present or not (Figure 1a). Numerous studies using mouse and human cells have investigated the effect of LPS on cell proliferation [59] and cytokine production by lymphocytes [60]. LPS has been noted to both activate [61] and inhibit [62] lymphocytic activation. However, most of the studies have shown that the mechanism of T cell activation by LPS is mediated by innate cells, such as monocytes or APCs, providing costimulatory molecules signals via direct cell contact [61,63]. Therefore, our results suggested that LPS may not trigger appropriate responses by M. natalensis APCs that support T cell proliferation in our model. However, the lack of specific antibodies and immunology tools to M. natalensis currently does not allow us to confirm the role of LPS on M. natalensis APCs or T cells. In contrast, M. natalensis splenic lymphocytes stimulated with LPS for 24 h increased secretion of TNF-α and IFN-γ which was also independent of IL-2 ( Figure 2a). This may be explained in part by differences of transcription, translation, protein processing, export, and protein degradation by each species of rodent [64]. Guy et al. also demonstrated that the large number of immunoreceptor tyrosine activation motifs (ITAM) within the T cell receptor (TCR)-CD3 complex (TCR-CD3 ITAM) play an important role in T cell development and function. Indeed, they have shown that low CD3 ITAM engage TCR-driven pathways that lead to cytokine production while high TCR-CD3 ITAM multiplicity promote T cell proliferation. These results support that proliferation and cytokine production can be two distinct events in T cells, dependent on the TCR signaling [65].
We also demonstrated that M. natalensis splenocytes stimulated with PHA require the presence of IL-2 to proliferate, while PHA efficiently induce C57BL/6J splenic lymphocytes proliferation without IL-2 cytokine (Figure 1b). Regardless of the presence/absence of IL-2, PHA was efficient to induce secretion of TNF-α but not IFN-γ by M. natalensis CD3 + T cells. However, IL-2 significantly decreased the production of both cytokines by C57BL/6J cells (Figure 2b). The decrease of IFN-γ and TNF-α secretion by C57BL/6J splenic lymphocytes stimulated with PHA in the presence of IL-2 suggests that exogenously added IL-2 may have induced T cell exhaustion resulting in functional impairment of T cells to secrete IFN-γ and TNF-α. Further studies need to confirm this hypothesis. We hypothesize that PHA combined with IL-2 may induce IL-2 receptor expression by T cells [45], resulting in enhanced proliferation by M. natalensis cells. However, lack of specific antibodies against M. natalensis do not allow us to confirm the effect of PHA on IL-2 receptor at this time. Finally, we demonstrated that ConA was sufficient to stimulate M. natalensis T cell proliferation and differentiation into effector T cells in the absence of IL-2 (Figures 1 and 3) and significantly increased the secretion of TNF-α and IFN-γ from M. natalensis splenic lymphocytes independent of the presence or absence of IL-2 ( Figure 2c). This suggests that ConA by itself triggered cross-linking of the TCR complex which leads to T cell proliferation and cytokine secretion among M. natalensis splenic cells, contrary to LPS and PHA mitogens [42]. Still, the molecular mechanisms by which TNF-α and IFN-γ genes expression and secretion occurs in response to each mitogen remain to be elucidated.

Conclusions
To conclude, only ConA was a strong stimulator of proliferation and differentiation into effector cells of M. natalensis CD3 + T cells. Thus, ConA-stimulating assays will allow us to determine ranges for IFN-γ and TNF-α in response to mitogen stimulation for M. natalensis-derived cells by flow cytometry (intracellular staining and CBA). These assays will be used to characterize the immune response in M. natalensis against infection and we believe this understanding of differences in distinct immune responses provides a critical underpinning for future studies on the immune response to pathogen infection in an increasingly important reservoir species. This is an important first step in the development of assays designed to understand the role of the innate and adaptive immune responses in this important reservoir species. Moving forward, there is a need to identify and optimize more immunology tools for M. natalensis, such as assays assessing cytotoxicity of CD4/CD8 T cells in vivo and in vitro.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request f, rom the corresponding author.