Neutrophil kinetics of Shigella infection in Macaca mulatta (Rhesus macaques)

Abstract

Introduction:

Neutrophil migration is a hallmark of acute inflammation and represents a key component of the early innate immune response to bacterial infection. We previously demonstrated the ability to track neutrophil movement from bone marrow to blood and subsequently to tissues using 5-bromo-2’-deoxyuridine (BrdU) pulse labeling in healthy adult rhesus macaques (Macaca mulatta), observing a consistent pattern of neutrophil kinetics during homeostasis.

Methods:

In this study, we extend our analyses to investigate the kinetics of neutrophil movement during acute inflammatory responses using Shigella sonnei, the causative agent of bacillary dysentery, as a model for acute bacterial infection. Twelve adult rhesus macaques were divided into three groups and challenged with varying doses of S. sonnei via gastric-oral lavage.

Results:

BrdU labeling revealed significant neutrophil consumption from blood into tissues and replenishment from bone marrow to blood as early as three days post-challenge, with a transient increase in neutrophil counts in blood at day nine post-challenge. Moreover, the extent of neutrophil kinetic changes correlated with the doses of S. sonnei.

Discussion:

These findings suggest that BrdU-labeled neutrophil kinetics provide valuable insights into neutrophil dynamics in vivo. This knowledge can be crucial for future studies that monitor acute inflammatory responses to infectious agents using nonhuman primate models. Additionally, it may aid in understanding the mechanisms of disease development and in creating effective intervention strategies.

Introduction

Neutrophils, the most abundant leukocytes in human circulation, are traditionally seen as key players in acute inflammatory responses. Due to their vital immune functions and short lifespan, they require continuous production in the bone marrow, a process known as granulopoiesis (Soehnlein et al., 2017). Neutrophil count is a critical hematological parameter for assessing acute inflammation, with both neutropenia and neutrophilia indicating various inflammatory conditions (Honda et al., 2016). However, interpreting absolute neutrophil counts (ANC) is complex due to the dynamic balance between neutrophil production in the bone marrow, migration between circulating and marginal pools, and clearance or apoptosis in tissues. Consequently, neutrophil counts often fluctuate, complicating clinical interpretations and underscoring the need for a deeper understanding of neutrophil roles and kinetics during acute and chronic inflammation (Margraf et al., 2022). Studying neutrophil kinetics in humans presents significant challenges due to both ethical and practical constraints. Nonhuman primates (NHPs), like rhesus macaques, share close genetic and physiological similarities with humans, making them invaluable models for researching human diseases and immune system functions.

Shigella spp. are Gram-negative intracellular bacteria that are transmitted via the fecal-oral route, causing bacillary dysentery in both humans and NHPs. Shigella represents a significant global health concern, particularly affecting young children under the age of five. Among the four species – S. sonnei, S. boydii, S. dysenteriae, and S. flexneri - S. flexneri and S. sonnei are the primary species responsible for endemic shigellosis, with S. flexneri being the most common species isolated from captive NHPs (Maxwell et al., 2022). Symptoms range from severe, life-threatening dysentery to asymptomatic carrier states (Maxwell et al., 2022). NHPs, such as rhesus macaques, are the only animal models that closely replicate Shigella infection as seen in human disease. When orally infected with S. flexneri, these primates develop symptoms of diarrhea and intestinal damage (Alphonse and Odendall, 2023).

Neutrophils play a crucial role in both host immunity and the pathogenesis of Shigella infection. In humans and rhesus macaques, shigellosis is characterized by significant neutrophil infiltration in the gut, which destabilizes gut integrity (Philpott et al., 2000; Islam et al., 2016). Upon entry through microfold cells into the gut submucosa, Shigella bacteria are phagocytosed by macrophages, which subsequently undergo apoptosis and release IL-1β to induce inflammation. Infected epithelial cells secrete IL-8, attracting neutrophils to the local tissues (Philpott et al., 2000). Emergency granulopoiesis, marked by increased bone marrow hematopoietic production to compensate for neutrophil depletion due to microbial infections, has been observed in animal models and human clinical studies (Manz and Boettcher, 2014). In a zebrafish larvae Shigella infection model, granulopoiesis plays critical protective roles (Willis et al., 2018). However, quantifying granulopoiesis remains challenging, with ANC used as a proximate estimation (Harrold et al., 2020).

5-bromo-2’-deoxyuridine (BrdU), a thymidine analog, incorporates into hematopoietic progenitor cells in the bone marrow, serving as a tool to characterize myeloid lineage cell differentiation in vivo. In earlier studies, we demonstrated that in vivo BrdU pulse-chase experiments could monitor changes in blood monocyte turnover rates, identify newly divided neutrophils in the bone marrow, and track their presence in blood circulation and tissues during homeostasis, aging, and viral and bacterial infections in rhesus macaques (Hasegawa et al., 2009; Mehra et al., 2012; Sugimoto et al., 2015; He et al., 2018; He et al., 2021). We observed that neutrophil kinetics were consistently and tightly regulated during homeostasis in adult rhesus macaques (He et al., 2018).

In the present study, we investigated neutrophil kinetics using in vivo BrdU pulse-chase labeling to better understand the host response and neutrophil movement during acute bacterial exposure, using Shigella infection in rhesus macaques as an experimental model. Rhesus macaques frequently harbor circulating antibodies to S. flexneri, as they are natural carriers (Sestak et al., 2003). Therefore, to avoid a cross-reactive immune response from any naturally acquired immunity to previous S. flexneri exposure or infection, S. sonnei was used as the infecting bacterial species. In this disease modeling effort, gastric-oral gavage exposure of S. sonnei was performed in rhesus macaques to induce clinical shigellosis. To further elucidate the host response, in addition to clinical signs (e.g., diarrhea), peripheral neutrophil counts and corresponding kinetics were defined using timed administration of BrdU.

Materials and methods

Rhesus macaques

Twelve Indian-origin young adult rhesus macaques (2 females and 10 males) aged between 6.9 to 15.3 years from the Tulane National Biomedical Research Center were used to study neutrophil kinetics following Shigella inoculation (Supplementary Table 1). The animals were specific pathogen-free of SIV, Simian Betaretrovirus (formerly known as Type D Simian Retrovirus), Macacine herpesvirus 1 (i.e. herpes B virus), and Simian T-cell Leukemia Virus type 1 (STLV-1). Since Shigella occurs naturally in primates, a pool of 60 animals was initially prescreened for existing S. flexneri and S. sonnei LPS serum IgG antibodies. An arbitrary 1:40 endpoint cutoff was used as inclusion criteria for this study. These animals were in excellent health and their stool cultures were negative for Shigella. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Tulane University Institutional Animal Care and Use Committee (National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011).

Shigella challenge

The S. sonnei strain WRAIR I Virulent (BEI NR-519) was used for all challenges within this study. Prior to S. sonnei inoculation, animals were fasted for ~16 hours and stomach acidity were neutralized with sodium bicarbonate administered by gastric-oral gavage. An inoculum of cultured S. sonnei organisms was delivered intragastrically in 20 mL of sterile saline at different doses into three groups of rhesus macaques (day 0; n = 4 for each group: Group 1 received 1 ×10¹¹ CFU, Group 2 received 2×10¹⁰ CFU, and Group 3 received 1.5×10¹⁰ CFU) (Figure 1). These inoculum doses were chosen as to align with prior Shigella studies in nonhuman primates (Labrec et al., 1964; Shipley et al., 2010). Shigella- induced diarrhea was quantified using a modified Bristol scale, in accordance with the following parameters: daily stool collections were analyzed for the presence of occult blood and cultured in Hektoen, MacConkey, and TSI agar to determine shedding of Shigella (Supplementary Table 2). We attempted to control for age-related changes in neutrophil kinetics by creating groups with a mixture of ages.

Figure 1

BrdU administration and blood collection

For neutrophil kinetics studies, the thymidine analogue BrdU (catalog number B5002-100G; Sigma-Aldrich, St. Louis, MO,USA) was prepared at 30 mg/ml in endotoxin-free PBS (catalog number TMS-012-A; EMD Millipore, Burlington, MA, USA), filter sterilized through a 0.2 µm polyethersulfone membrane (Steriflip, catalog number SCGP00525 or 09-740-2A; ThermoFisher Scientific, Waltham, MA, USA), and administered intravenously at a dose of 60 mg/kg body weight into each rhesus macaque on day -1 pre-Shigella challenge. Blood samples were collected with EDTA anticoagulant for BrdU staining, flow cytometry, and hematology analyses on days 1, 4, 7, 10, and 14 after initial BrdU administration (Figure 1).

Flow cytometry and hematology analyses

Cellular immunophenotyping and staining for BrdU incorporation were performed as previously described (Sugimoto et al., 2015; He et al., 2018; He et al., 2021). Briefly, 200 µl of EDTA–anticoagulated whole blood were washed with PBS and stained with surface monoclonal antibodies (Supplementary Table 3). Red blood cells were lysed with FACS lysing solution (catalog number 349202; BD Biosciences, San Jose, CA, USA), and remaining cells were permeabilized using a three-step Cytofix/Cytoperm protocol per the manufacturer’s instructions (BD Biosciences). For analysis of BrdU incorporation, cells were incubated with DNase I (catalog number DN25, Sigma-Aldrich) at 37°for 1 hour and then stained with anti-BrdU antibody for 20 minutes at room temperature. After washing, cells were fixed in 250 µL of 1% paraformaldehyde in PBS. Samples were acquired with a LSRFortessa flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (version 10; FlowJo, LLC, Ashland, OR, USA). Hematology analyses were performed on a Sysmex XT-2000iV automated hematology analyzer (Sysmex America, Lincolnshire, IL). Absolute cell counts for neutrophils were directly measured from the automated hematology analyzer. Gating for neutrophils was performed as previously demonstrated (He et al., 2018). Briefly, granulocytes were first gated from intermediate to high forward scatter (FSC) and side scatter (SSC) fractions and then separated based on the expression of HLA-DR, with neutrophils negative for CD123 (FSC/SSC^high/dim, HLA-DR⁻, CD3⁻, CD20⁻, CD123⁻). Neutrophil content was confirmed via myeloperoxidase expression.

Statistical analyses

The nonparamtric Friedman test and post-analysis comparisons were performed on neutrophil counts at different days pre- and post-Shigella challenge. Kruskal-Wallis analyses were used to compare results between different dosage groups. We applied a previously established neutrophil kinetic model, derived from healthy adult macaques (He et al., 2018), to fit the kinetics following Shigella challenge and estimated the time at which BrdU-labeled neutrophils peak in circulation. Graphs were prepared using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). A p value <0.05 was considered statistically significant.

Results

Neutrophil kinetics shift in response to acute Shigella infection

To investigate the effects of acute Shigella infection on neutrophil kinetics, we monitored neutrophil numbers using complete blood cell counts and assessed kinetics by immunophenotyping and flow cytometry staining of BrdU-labeled neutrophils. We administered BrdU (60 mg/kg body weight) intravenously to 12 young adult rhesus macaques (n = 4 per group) on day -1, and then challenged them with a single dose of different concentrations of S. sonnei (Group 1: 1 ×10¹¹ CFU; Group 2: 2×10¹⁰ CFU; Group 3: 1.5×10¹⁰ CFU) orally on day 0 (Figure 1). Animals exposed at all dose levels developed clinical shigellosis, with diarrheal indexes exceeding 3.0 (Supplementary Table 2). Animals tested positive for S. sonnei in fecal cultures 2 days post-infection and remained positive for at least 7 days post-infection, with those in the lower dose group (1.5×10¹⁰ CFU) resolving quicker than those in the highest dose group (1 ×10¹¹ CFU) (Supplementary Table 2). Whole blood samples were collected on days 0 (pre-challenge), 3, 6, 9, and 13 (days 1, 4, 7, 10, and 14 after BrdU administration), stained, and analyzed for immunophenotyping and BrdU incorporation. On day 20, a second injection of BrdU (60 mg/kg body weight) was administered intravenously, and blood samples were collected on days 21, 24, and 26 (days 1, 4, and 7 after second BrdU administration) for immunophenotyping and BrdU incorporation analysis (Figure 1). Neutrophil counts decreased slightly but not significantly at day 3 and recovered to pre-challenge levels by day 6 post-inoculation. Interestingly, on day 9 post-inoculation, neutrophil counts were significantly higher than pre-challenge and day 3 post-challenge (Figure 2A). In all three Shigella-exposed groups, transient neutrophilia occurred on day 9 post-inoculation and quickly resolved, reaching normal neutrophil ranges by day 13 (Figure 2B).

Figure 2

BrdU was administered a day before S. sonnei inoculation to label the bone marrow dividing neutrophil precursors under undisturbed conditions and to investigate the movement of neutrophils in vivo after S. sonnei inoculation. On day 4 post-BrdU injection (3 days post-infection), we observed significantly higher percentages of BrdU-labeled neutrophils in blood circulation in all 12 Shigella-inoculated animals compared to uninfected healthy macaques, suggesting that newly divided neutrophils mobilized from the bone marrow into circulation in response to Shigella inoculation in the early stages of the disease (Figure 3A). Neutrophil kinetics were modeled as previously described, and the time post-BrdU injection was calculated for each animal to reach peak BrdU-labeled neutrophil levels in blood (dashed lines, Figures 3C–E) (Hasegawa et al., 2009). Results indicate that the timing of peak BrdU-labeled neutrophils is highly dependent on the Shigella inoculating dose; neutrophils from the highest Shigella inoculation dose group were released to blood stream the earliest (Figure 3B).

Figure 3

Because BrdU-labeled neutrophils were cleared from the system by about two weeks, BrdU was administered again 20 days after the initial bacterial inoculation to monitor neutrophil kinetics in the infection resolution phase, during which fecal cultures for all animals were negative for Shigella (Supplementary Table 2). Neutrophil kinetics had reestablished to homeostatic levels in all three groups of animals, characterized by low BrdU-labeled neutrophil percentages on day 4 and higher BrdU-labeled neutrophil percentages on day 7 following the second BrdU administration, as also observed in healthy uninoculated animals (Figure 4).

Figure 4

Discussion

Neutrophils are the primary myeloid and white blood cell (WBC) component in blood of humans and nonhuman primates (NHPs). These short-lived, first-responder immune cells patrol the body and rapidly respond to stimuli, infections, or tissue damage by eliminating pathogens and mediating acute inflammation, actions which necessitate constant replenishment from the bone marrow. Neutrophil infiltration, characterized by their movement into effector sites, is a hallmark of acute inflammation. Our research, along with others, has demonstrated that the bone marrow continuously produces large quantities of neutrophils for release into the bloodstream throughout an individual’s lifespan (Price et al., 1996; He et al., 2018; He et al., 2021). Although neutrophil counts are commonly used as a clinical parameter, the in vivo movement of neutrophils during acute infections is not always accurately reflected by blood cell counts. Determining the lifespan of myeloid cells in humans is challenging due to the potential toxicity of in vivo labeling agents and the difficulties associated with performing repeated bone marrow and blood sampling required to confirm cell kinetics. Therefore, NHPs are valuable models for in vivo cell proliferation labeling due to their genetic and physiological similarities to humans, as well as their comparable WBC composition (Sugimoto et al., 2015; Didier et al., 2016). Consequently, NHPs serve as important animal models for gaining a better understanding of the human immune system.

In this study, we utilized in vivo labeling with the thymidine analog BrdU to examine neutrophil development and kinetics in rhesus macaques during acute inflammation induced by S. sonnei infection. When administered, BrdU is incorporated as a thymidine analog by dividing hematopoietic stem cells in the bone marrow during DNA synthesis in the S-phase of the cell cycle, making it a reliable marker for identifying dividing cells. Shigella spp. are frequently encountered as enteric pathogens in captive NHPs (Sestak et al., 2003). However, the response of neutrophils to a self-resolving S. sonnei infection in rhesus macaques during the acute phase remained undefined. Accordingly, the kinetics of BrdU incorporation into neutrophils was tracked using the sampling strategy outlined in Figure 1. Shigella infection represents a local bacterial infection where neutrophils quickly mobilize to the local tissue, presumably the gut, to help elicit the host response to the infection. Relatively high doses of S. sonnei were selected as NHPs are generally resistant to clinical infection with Shigella spp., in contrast to humans, where the infectious dose is estimated to be as low as 10–100 organisms (DuPont et al., 1989). These data rely on the common assumption that pre-infection bone marrow proliferation are comparable across our groups, with an expected expansion and increase in turnover after bacterial challenge. Our findings document the development of neutrophils as they migrate from the bone marrow to the blood and subsequently transition into tissues in response to Shigella infection, though we did not directly measure the tissue migration.

We observed a transient neutrophilia at 9 days post-S. sonnei challenge in all three groups of rhesus macaques (Figure 2A). However, the kinetics, measured by the percentage of BrdU-labeled neutrophils in circulation, indicated significant movement to tissues and replenishment from the bone marrow reserve as early as 3 days post-challenge (Figure 3A). The neutrophilia observed at day nine post-S. sonnei challenge reflected compensatory neutrophil production in the bone marrow, while the actual neutrophil response to the infection occurred earlier (day three), as evidenced by the mobilization of neutrophils from the bone marrow reservoir (Figure 3B).

Our previous research demonstrated that in healthy rhesus macaques, BrdU-labeled neutrophils remain in the bone marrow for 4–5 days before being released and are cleared from the blood approximately 14 days after BrdU administration (He et al., 2018). Since BrdU was injected before the Shigella inoculation and cleared from the system quickly, and because neutrophil kinetics are consistent among young adult rhesus macaques during homeostasis, we assumed that neutrophil proliferation in the bone marrow at day -1 before inoculation, as reflected by peaks of BrdU-labeled neutrophils in the blood later on, was similar across all groups. This assumption allowed us to estimate the transit time for BrdU-labeled neutrophils to reach their peak levels in the blood in each group of animals (Figures 3B–E). Interestingly, the magnitude of the kinetics shift and the earlier release of neutrophils from the bone marrow directly correlated with S. sonnei inoculating dose, indicating that the movement of neutrophils reflected the intensity of the host response and the consumption of blood neutrophils post-challenge, possibly in a dose-dependent manner (Figures 3C–E). These alterations in neutrophil kinetics quickly returned to the baseline in the animals once the Shigella infections resolved, as indicated by negative fecal cultures (Figure 4; Supplementary Table 2).

In summary, our study demonstrates that neutrophil kinetics, measured by BrdU pulse-chase labeling, provide a valuable tool for monitoring neutrophil movement in vivo in rhesus macaques. During homeostasis, neutrophil kinetics are highly regulated, with significant bone marrow production observed (He et al., 2018). In response to acute infection, neutrophils are released earlier from the bone marrow reserve into the circulation, reflecting the intensity of the host response and the bacterial burden (Figures 3A, B). This movement, which correlates with inoculation doses, can be sensitively measured by BrdU kinetics and may serve as a useful metric for challenge model and any subsequent vaccine or therapeutic efficacy studies. Our findings also show that neutrophil kinetics return to baseline once Shigella infection resolves (Figure 4). These results emphasize the utility of NHP models in studying the kinetics of myeloid lineage cells and the pathogenesis of infectious diseases in humans. In addition, we demonstrate that kinetic shifts within neutrophils in response to infection are not fully encapsulated by purely measuring neutrophil counts. Consequently, in vivo BrdU pulse-chase studies are invaluable for understanding the development, kinetics, and turnover of replicating immune cells, enhancing our knowledge of innate immune responses to infectious pathogens and the role of myeloid cells in infections and inflammation. Although we focused purely upon the neutrophil response, other innate immune mechanisms may be modulated by, or important in the control of, Shigella infection. Future studies should focus on understanding of other responses as well.

References

• 1

  AlphonseN.OdendallC. (2023). Animal models of shigellosis: a historical overview. Curr. Opin. Immunol.85, 102399. doi: 10.1016/j.coi.2023.102399

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 2

  DidierE. S.MacLeanA. G.MohanM.DidierP. J.LacknerA. A.KurodaM. J. (2016). Contributions of nonhuman primates to research on aging. Vet. Pathol.53, 277–290. doi: 10.1177/0300985815622974

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 3

  DuPontH. L.LevineM. M.HornickR. B.FormalS. B. (1989). Inoculum size in shigellosis and implications for expected mode of transmission. J. Infect. Dis.159, 1126–1128. doi: 10.1093/infdis/159.6.1126

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 4

  HarroldJ.Olsson GisleskogP.DelorI.JacqminP.Perez-RuixoJ. J.NarayananA.et al. (2020). Quantification of radiation injury on neutropenia and the link between absolute neutrophil count time course and overall survival in nonhuman primates treated with G-CSF. Pharm. Res.37, 102. doi: 10.1007/s11095-020-02839-3

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 5

  HasegawaA.LiuH.LingB.BordaJ. T.AlvarezX.SugimotoC.et al. (2009). The level of monocyte turnover predicts disease progression in the macaque model of AIDS. Blood114, 2917–2925. doi: 10.1182/blood-2009-02-204263

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 6

  HeZ.AllersC.SugimotoC.AhmedN.FujiokaH.KimW. K.et al. (2018). Rapid turnover and high production rate of myeloid cells in adult rhesus macaques with compensations during aging. J. Immunol.200, 4059–4067. doi: 10.4049/jimmunol.1800207

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 7

  HeZ.FahlbergM. D.TakahashiN.SlisarenkoN.RoutN.DidierE. S.et al. (2021). Declining neutrophil production despite increasing G-CSF levels is associated with chronic inflammation in elderly rhesus macaques. J. Leukoc. Biol.109, 1033–1043. doi: 10.1002/jlb.1hi1120-779r

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 8

  HondaT.UeharaT.MatsumotoG.AraiS.SuganoM. (2016). Neutrophil left shift and white blood cell count as markers of bacterial infection. Clin. Chim. Acta457, 46–53. doi: 10.1016/j.cca.2016.03.017

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 9

  IslamD.LombardiniE.RuamsapN.ImerbsinR.KhantapuraP.TeoI.et al. (2016). Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist. Immunology147, 178–189. doi: 10.1111/imm.12549

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 10

  LabrecE. H.SchneiderH.MagnaniT. J.FormalS. B. (1964). Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J. Bacteriol.88, 1503–1518. doi: 10.1128/jb.88.5.1503-1518.1964

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 11

  ManzM. G.BoettcherS. (2014). Emergency granulopoiesis. Nat. Rev. Immunol.14, 302–314. doi: 10.1038/nri3660

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 12

  MargrafA.LowellC. A.ZarbockA. (2022). Neutrophils in acute inflammation: current concepts and translational implications. Blood139, 2130–2144. doi: 10.1182/blood.2021012295

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 13

  MaxwellA. R.HerrodJ. A.HutchinsonE. K. (2022). A comparison of different antibiotic regimens for the treatment of naturally acquired shigellosis in rhesus and pigtailed macaques (Macaca mulatta and nemestrina). J. Med. Primatol.51, 374–380. doi: 10.1111/jmp.12608

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 14

  MehraS.GoldenN. A.StuckeyK.DidierP. J.DoyleL. A.Russell-LodrigueK. E.et al. (2012). The Mycobacterium tuberculosis stress response factor SigH is required for bacterial burden as well as immunopathology in primate lungs. J. Infect. Dis.205, 1203–1213. doi: 10.1093/infdis/jis102

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 15

  National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. (2011). Guide for the Care and Use of Laboratory Animals: Eighth Edition (Washington (DC): The National Academies Press). Available online at: https://www.ncbi.nlm.nih.gov/books/NBK54050/ doi:10.17226/12910.

  • Google Scholar
  • View reference in article

• 16

  PhilpottD. J.EdgeworthJ. D.SansonettiP. J. (2000). The pathogenesis of Shigella flexneri infection: lessons from in vitro and in vivo studies. Philos. Trans. R. Soc Lond. B. Biol. Sci.355, 575–586. doi: 10.1098/rstb.2000.0599

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 17

  PriceT. H.ChattaG. S.DaleD. C. (1996). Effect of recombinant granulocyte colony-stimulating factor on neutrophil kinetics in normal young and elderly humans. Blood88, 335–340. doi: 10.1182/blood.v88.1.335.335

  • CrossRef
  • Google Scholar

• 18

  SestakK.MerrittC. K.BordaJ.SaylorE.SchwambergerS. R.CogswellF.et al. (2003). Infectious agent and immune response characteristics of chronic enterocolitis in captive rhesus macaques. Infect. Immun.71, 4079–4086. doi: 10.1128/iai.71.7.4079-4086.2003

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 19

  ShipleyS. T.PandaA.KhanA. Q.KrielE. H.MacielM.LivioS.et al. (2010). A challenge model for Shigella dysenteriae 1 in cynomolgus monkeys (Macaca fascicularis). Comp. Med.60, 54–61.

  • Google Scholar
  • View reference in article

• 20

  SoehnleinO.SteffensS.HidalgoA.WeberC. (2017). Neutrophils as protagonists and targets in chronic inflammation. Nat. Rev. Immunol.17, 248–261. doi: 10.1038/nri.2017.10

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 21

  SugimotoC.HasegawaA.SaitoY.FukuyoY.ChiuK. B.CaiY.et al. (2015). Differentiation kinetics of blood monocytes and dendritic cells in macaques: insights to understanding human myeloid cell development. J. Immunol.195, 1774–1781. doi: 10.4049/jimmunol.1500522

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 22

  WillisA. R.TorracaV.GomesM. C.ShelleyJ.Mazon-MoyaM.FillouxA.et al. (2018). Shigella-induced emergency granulopoiesis protects zebrafish larvae from secondary infection. mBio9. doi: 10.1128/mBio.00933-18

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article