Dissemination of Chlamydia from the reproductive tract to the gastro-intestinal tract occurs in stages and relies on Chlamydia transport by host cells

English version

Autoři: Savannah E. Howe ^aff001; Nita Shillova ^aff001; Vjollca Konjufca ^aff001
Působiště autorů: Department of Microbiology, Southern Illinois University, Carbondale, Illinois, United States of America ^aff001
Vyšlo v časopise: Dissemination of Chlamydia from the reproductive tract to the gastro-intestinal tract occurs in stages and relies on Chlamydia transport by host cells. PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1008207
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008207

Souhrn

Chlamydia trachomatis is a Gram-negative bacterial pathogen and a major cause of sexually transmitted disease and preventable blindness. In women, infections with C. trachomatis may lead to pelvic inflammatory disease (PID), ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting the female reproductive tract (FRT), Chlamydia spp. are routinely found in the gastro-intestinal (GI) tract of animals and humans and can be a reservoir for reinfection of the FRT. Whether Chlamydia disseminates from the FRT to the GI tract via internal routes remains unknown. Using mouse-specific C. muridarum as a model pathogen we show that Chlamydia disseminates from the FRT to the GI tract in a stepwise manner, by first infecting the FRT-draining iliac lymph nodes (ILNs), then the spleen, then the GI tract. Tissue CD11c⁺ DCs mediate the first step: FRT to ILN Chlamydia transport, which relies on CCR7:CCL21/CCL19 signaling. The second step, Chlamydia transport from ILN to the spleen, also relies on cell transport. However, this step is dependent on cell migration mediated by sphingosine 1-phosphate (S1P) signaling. Finally, spleen to GI tract Chlamydia spread is the third critical step, and is significantly hindered in splenectomized mice. Inhibition of Chlamydia dissemination significantly reduces or precludes the induction of Chlamydia-specific serum IgG antibodies, presence of which is correlated with FRT pathology in women. This study reveals important insights in context of Chlamydia spp. pathogenesis and will inform the development of therapeutic targets and vaccines to combat this pathogen.

Klíčová slova:

Cell migration – Genitourinary infections – Chlamydia – Chlamydia infection – Lymph nodes – Spleen – T cells

Zdroje

1. WHO. Sexually transmitted infections (STIs) https://www.who.int/en/news-room/fact-sheets/detail/sexually-transmitted-infections-(stis): WHO; 2019 [cited 2019 06.16.2019]. Available from: https://www.who.int/en/news-room/fact-sheets/detail/sexually-transmitted-infections-(stis).

2. CDC. Reported STDs in the United States, 2017. https://www.cdc.gov/nchhstp/newsroom/docs/factsheets/std-trends-508.pdf: CDC; 2018. p. 3.

3. Haggerty CL, Gottlieb SL, Taylor BD, Low N, Xu F, Ness RB. Risk of sequelae after Chlamydia trachomatis genital infection in women. J Infect Dis. 2010;201 Suppl 2:S134–55. Epub 2010/05/28. doi: 10.1086/652395 20470050.

4. Oakeshott P, Kerry S, Aghaizu A, Atherton H, Hay S, Taylor-Robinson D, et al. Randomised controlled trial of screening for Chlamydia trachomatis to prevent pelvic inflammatory disease: the POPI (prevention of pelvic infection) trial. BMJ. 2010;340:c1642. Epub 2010/04/10. doi: 10.1136/bmj.c1642 20378636; PubMed Central PMCID: PMC2851939.

5. Price MJ, Ades AE, De Angelis D, Welton NJ, Macleod J, Soldan K, et al. Risk of pelvic inflammatory disease following Chlamydia trachomatis infection: analysis of prospective studies with a multistate model. Am J Epidemiol. 2013;178(3):484–92. Epub 2013/07/03. doi: 10.1093/aje/kws583 23813703; PubMed Central PMCID: PMC3727337.

6. Brunham RC, Gottlieb SL, Paavonen J. Pelvic inflammatory disease. N Engl J Med. 2015;372(21):2039–48. Epub 2015/05/21. doi: 10.1056/NEJMra1411426 25992748.

7. Madeleine MM, Anttila T, Schwartz SM, Saikku P, Leinonen M, Carter JJ, et al. Risk of cervical cancer associated with Chlamydia trachomatis antibodies by histology, HPV type and HPV cofactors. Int J Cancer. 2007;120(3):650–5. Epub 2006/11/11. doi: 10.1002/ijc.22325 17096345.

8. Schust DJ, Ibana JA, Buckner LR, Ficarra M, Sugimoto J, Amedee AM, et al. Potential Mechanisms for Increased HIV-1 Transmission Across the Endocervical Epithelium During C. trachomatis Infection. Curr HIV Res. 2012;10(3):218–27. Epub 2012/03/06. CHIVR-EPUB-20120229-003 [pii]. doi: 10.2174/157016212800618093 22384841.

9. Moulder JW. Interaction of chlamydiae and host cells in vitro. Microbiol Rev. 1991;55(1):143–90. Epub 1991/03/01. 2030670; PubMed Central PMCID: PMC372804.

10. Read TD, Brunham RC, Shen C, Gill SR, Heidelberg JF, White O, et al. Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res. 2000;28(6):1397–406. Epub 2000/02/24. doi: 10.1093/nar/28.6.1397 10684935; PubMed Central PMCID: PMC111046.

11. de la Maza LM, Pal S, Khamesipour A, Peterson EM. Intravaginal inoculation of mice with the Chlamydia trachomatis mouse pneumonitis biovar results in infertility. Infect Immun. 1994;62(5):2094–7. Epub 1994/05/01. 8168974; PubMed Central PMCID: PMC186471.

12. Shah AA, Schripsema JH, Imtiaz MT, Sigar IM, Kasimos J, Matos PG, et al. Histopathologic changes related to fibrotic oviduct occlusion after genital tract infection of mice with Chlamydia muridarum. Sex Transm Dis. 2005;32(1):49–56. Epub 2004/12/23. doi: 10.1097/01.olq.0000148299.14513.11 15614121.

13. Budrys NM, Gong S, Rodgers AK, Wang J, Louden C, Shain R, et al. Chlamydia trachomatis antigens recognized in women with tubal factor infertility, normal fertility, and acute infection. Obstet Gynecol. 2012;119(5):1009–16. Epub 2012/04/25. doi: 10.1097/AOG.0b013e3182519326 22525912; PubMed Central PMCID: PMC4608258.

14. Rodgers AK, Wang J, Zhang Y, Holden A, Berryhill B, Budrys NM, et al. Association of tubal factor infertility with elevated antibodies to Chlamydia trachomatis caseinolytic protease P. Am J Obstet Gynecol. 2010;203(5):494 e7–e14. Epub 2010/07/21. doi: 10.1016/j.ajog.2010.06.005 20643392; PubMed Central PMCID: PMC3223063.

15. Sturdevant GL, Caldwell HD. Innate immunity is sufficient for the clearance of Chlamydia trachomatis from the female mouse genital tract. Pathog Dis. 2014;72(1):70–3. Epub 2014/03/04. doi: 10.1111/2049-632X.12164 24585717; PubMed Central PMCID: PMC4152394.

16. Carmichael JR, Tifrea D, Pal S, de la Maza LM. Differences in infectivity and induction of infertility: a comparative study of Chlamydia trachomatis strains in the murine model. Microbes Infect. 2013;15(3):219–29. Epub 2013/01/05. doi: 10.1016/j.micinf.2012.12.001 23287699; PubMed Central PMCID: PMC3602122.

17. Rank RG, Yeruva L. Hidden in plain sight: chlamydial gastrointestinal infection and its relevance to persistence in human genital infection. Infect Immun. 2014;82(4):1362–71. Epub 2014/01/15. doi: 10.1128/IAI.01244-13 24421044; PubMed Central PMCID: PMC3993372.

18. Rank RG, Yeruva L. An alternative scenario to explain rectal positivity in Chlamydia-infected individuals. Clin Infect Dis. 2015;60(10):1585–6. Epub 2015/02/05. doi: 10.1093/cid/civ079 25648236.

19. Yeruva L, Spencer N, Bowlin AK, Wang Y, Rank RG. Chlamydial infection of the gastrointestinal tract: a reservoir for persistent infection. Pathog Dis. 2013;68(3):88–95. Epub 2013/07/12. doi: 10.1111/2049-632X.12052 23843274; PubMed Central PMCID: PMC3751173.

20. Drummond F, Ryder N, Wand H, Guy R, Read P, McNulty AM, et al. Is azithromycin adequate treatment for asymptomatic rectal chlamydia? Int J STD AIDS. 2011;22(8):478–80. Epub 2011/07/12. doi: 10.1258/ijsa.2011.010490 21742812.

21. Hathorn E, Opie C, Goold P. What is the appropriate treatment for the management of rectal Chlamydia trachomatis in men and women? Sex Transm Infect. 2012;88(5):352–4. Epub 2012/04/21. doi: 10.1136/sextrans-2011-050466 22517887.

22. Zhang Q, Huang Y, Gong S, Yang Z, Sun X, Schenken R, et al. In Vivo and Ex Vivo Imaging Reveals a Long-Lasting Chlamydial Infection in the Mouse Gastrointestinal Tract following Genital Tract Inoculation. Infect Immun. 2015;83(9):3568–77. Epub 2015/06/24. doi: 10.1128/IAI.00673-15 26099591; PubMed Central PMCID: PMC4534645.

23. Dai J, Zhang T, Wang L, Shao L, Zhu C, Zhang Y, et al. Intravenous Inoculation with Chlamydia muridarum Leads to a Long-Lasting Infection Restricted to the Gastrointestinal Tract. Infect Immun. 2016;84(8):2382–8. Epub 2016/06/09. doi: 10.1128/IAI.00432-16 27271744; PubMed Central PMCID: PMC4962645.

24. Joffre O, Nolte MA, Sporri R, Reis e Sousa C. Inflammatory signals in dendritic cell activation and the induction of adaptive immunity. Immunol Rev. 2009;227(1):234–47. Epub 2009/01/06. doi: 10.1111/j.1600-065X.2008.00718.x 19120488.

25. Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, et al. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity. 2004;21(2):279–88. Epub 2004/08/17. doi: 10.1016/j.immuni.2004.06.014 15308107.

26. Forster R, Davalos-Misslitz AC, Rot A. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev Immunol. 2008;8(5):362–71. Epub 2008/04/02. doi: 10.1038/nri2297 18379575.

27. Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell. 1999;99(1):23–33. Epub 1999/10/16. doi: 10.1016/s0092-8674(00)80059-8 10520991.

28. Weber M, Hauschild R, Schwarz J, Moussion C, de Vries I, Legler DF, et al. Interstitial dendritic cell guidance by haptotactic chemokine gradients. Science. 2013;339(6117):328–32. Epub 2013/01/19. doi: 10.1126/science.1228456 23329049.

29. Czeloth N, Bernhardt G, Hofmann F, Genth H, Forster R. Sphingosine-1-phosphate mediates migration of mature dendritic cells. J Immunol. 2005;175(5):2960–7. Epub 2005/08/24. doi: 10.4049/jimmunol.175.5.2960 16116182.

30. Brinkmann V, Cyster JG, Hla T. FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function. Am J Transplant. 2004;4(7):1019–25. Epub 2004/06/16. doi: 10.1111/j.1600-6143.2004.00476.x 15196057.

31. Brunham RC, Rey-Ladino J. Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine. Nat Rev Immunol. 2005;5(2):149–61. doi: 10.1038/nri1551 15688042.

32. MacDonald TT, Monteleone I, Fantini MC, Monteleone G. Regulation of homeostasis and inflammation in the intestine. Gastroenterology. 2011;140(6):1768–75. Epub 2011/05/03. doi: 10.1053/j.gastro.2011.02.047 21530743.

33. Rhodes JW, Tong O, Harman AN, Turville SG. Human Dendritic Cell Subsets, Ontogeny, and Impact on HIV Infection. Front Immunol. 2019;10:1088. Epub 2019/06/04. doi: 10.3389/fimmu.2019.01088 31156637; PubMed Central PMCID: PMC6532592.

34. Waisman A, Lukas D, Clausen BE, Yogev N. Dendritic cells as gatekeepers of tolerance. Semin Immunopathol. 2017;39(2):153–63. Epub 2016/07/28. doi: 10.1007/s00281-016-0583-z 27456849.

35. Shen R, Kappes JC, Smythies LE, Richter HE, Novak L, Smith PD. Vaginal myeloid dendritic cells transmit founder HIV-1. J Virol. 2014;88(13):7683–8. Epub 2014/04/18. doi: 10.1128/JVI.00766-14 24741097; PubMed Central PMCID: PMC4054437.

36. Shen R, Smythies LE, Clements RH, Novak L, Smith PD. Dendritic cells transmit HIV-1 through human small intestinal mucosa. J Leukoc Biol. 2010;87(4):663–70. Epub 2009/12/17. doi: 10.1189/jlb.0909605 20007245; PubMed Central PMCID: PMC2858307.

37. Nakamizo S, Egawa G, Tomura M, Sakai S, Tsuchiya S, Kitoh A, et al. Dermal Vgamma4(+) gammadelta T cells possess a migratory potency to the draining lymph nodes and modulate CD8(+) T-cell activity through TNF-alpha production. J Invest Dermatol. 2015;135(4):1007–15. Epub 2014/12/11. doi: 10.1038/jid.2014.516 25493651.

38. Jang MH, Sougawa N, Tanaka T, Hirata T, Hiroi T, Tohya K, et al. CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes. J Immunol. 2006;176(2):803–10. Epub 2006/01/06. doi: 10.4049/jimmunol.176.2.803 16393963.

39. Hintzen G, Ohl L, del Rio ML, Rodriguez-Barbosa JI, Pabst O, Kocks JR, et al. Induction of tolerance to innocuous inhaled antigen relies on a CCR7-dependent dendritic cell-mediated antigen transport to the bronchial lymph node. J Immunol. 2006;177(10):7346–54. Epub 2006/11/04. doi: 10.4049/jimmunol.177.10.7346 17082654.

40. Pham TH, Baluk P, Xu Y, Grigorova I, Bankovich AJ, Pappu R, et al. Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning. J Exp Med. 2010;207(1):17–27. Epub 2009/12/23. doi: 10.1084/jem.20091619 20026661; PubMed Central PMCID: PMC2812554.

41. Vaahtomeri K, Brown M, Hauschild R, De Vries I, Leithner AF, Mehling M, et al. Locally Triggered Release of the Chemokine CCL21 Promotes Dendritic Cell Transmigration across Lymphatic Endothelia. Cell Rep. 2017;19(5):902–9. Epub 2017/05/04. doi: 10.1016/j.celrep.2017.04.027 28467903; PubMed Central PMCID: PMC5437727.

42. Braun A, Worbs T, Moschovakis GL, Halle S, Hoffmann K, Bolter J, et al. Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration. Nat Immunol. 2011;12(9):879–87. Epub 2011/08/16. doi: 10.1038/ni.2085 21841786.

43. Clatworthy MR, Aronin CE, Mathews RJ, Morgan NY, Smith KG, Germain RN. Immune complexes stimulate CCR7-dependent dendritic cell migration to lymph nodes. Nat Med. 2014;20(12):1458–63. Epub 2014/11/11. doi: 10.1038/nm.3709 25384086; PubMed Central PMCID: PMC4283039.

44. Kabashima K, Shiraishi N, Sugita K, Mori T, Onoue A, Kobayashi M, et al. CXCL12-CXCR4 engagement is required for migration of cutaneous dendritic cells. Am J Pathol. 2007;171(4):1249–57. Epub 2007/09/08. doi: 10.2353/ajpath.2007.070225 17823289; PubMed Central PMCID: PMC1988874.

45. Johnson LA, Jackson DG. The chemokine CX3CL1 promotes trafficking of dendritic cells through inflamed lymphatics. J Cell Sci. 2013;126(Pt 22):5259–70. Epub 2013/09/06. doi: 10.1242/jcs.135343 24006262; PubMed Central PMCID: PMC3828594.

46. Qu C, Edwards EW, Tacke F, Angeli V, Llodra J, Sanchez-Schmitz G, et al. Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes. J Exp Med. 2004;200(10):1231–41. Epub 2004/11/10. doi: 10.1084/jem.20032152 15534368; PubMed Central PMCID: PMC2211916.

47. Vander Lugt B, Tubo NJ, Nizza ST, Boes M, Malissen B, Fuhlbrigge RC, et al. CCR7 plays no appreciable role in trafficking of central memory CD4 T cells to lymph nodes. J Immunol. 2013;191(6):3119–27. Epub 2013/08/13. doi: 10.4049/jimmunol.1200938 23935190; PubMed Central PMCID: PMC3784989.

48. Lindquist RL, Shakhar G, Dudziak D, Wardemann H, Eisenreich T, Dustin ML, et al. Visualizing dendritic cell networks in vivo. Nat Immunol. 2004;5(12):1243–50. Epub 2004/11/16. doi: 10.1038/ni1139 15543150.

49. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol. 2013;31:563–604. Epub 2013/03/23. doi: 10.1146/annurev-immunol-020711-074950 23516985; PubMed Central PMCID: PMC3853342.

50. Jenne CN, Enders A, Rivera R, Watson SR, Bankovich AJ, Pereira JP, et al. T-bet-dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow. J Exp Med. 2009;206(11):2469–81. Epub 2009/10/08. doi: 10.1084/jem.20090525 19808259; PubMed Central PMCID: PMC2768857.

51. Walzer T, Chiossone L, Chaix J, Calver A, Carozzo C, Garrigue-Antar L, et al. Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat Immunol. 2007;8(12):1337–44. Epub 2007/10/30. doi: 10.1038/ni1523 17965716.

52. Lewis ND, Haxhinasto SA, Anderson SM, Stefanopoulos DE, Fogal SE, Adusumalli P, et al. Circulating monocytes are reduced by sphingosine-1-phosphate receptor modulators independently of S1P3. J Immunol. 2013;190(7):3533–40. Epub 2013/02/26. doi: 10.4049/jimmunol.1201810 23436932.

53. McDonald D, Wu L, Bohks SM, KewalRamani VN, Unutmaz D, Hope TJ. Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science. 2003;300(5623):1295–7. Epub 2003/05/06. doi: 10.1126/science.1084238 12730499.

54. Cavrois M, Neidleman J, Kreisberg JF, Greene WC. In vitro derived dendritic cells trans-infect CD4 T cells primarily with surface-bound HIV-1 virions. PLoS Pathog. 2007;3(1):e4. Epub 2007/01/24. doi: 10.1371/journal.ppat.0030004 17238285; PubMed Central PMCID: PMC1779297.

55. Rodgers AK, Budrys NM, Gong S, Wang J, Holden A, Schenken RS, et al. Genome-wide identification of Chlamydia trachomatis antigens associated with tubal factor infertility. Fertil Steril. 2011;96(3):715–21. Epub 2011/07/12. doi: 10.1016/j.fertnstert.2011.06.021 21742324; PubMed Central PMCID: PMC3225487.

56. Sziller I, Fedorcsak P, Csapo Z, Szirmai K, Linhares IM, Papp Z, et al. Circulating antibodies to a conserved epitope of the Chlamydia trachomatis 60-kDa heat shock protein is associated with decreased spontaneous fertility rate in ectopic pregnant women treated by salpingectomy. Am J Reprod Immunol. 2008;59(2):99–104. Epub 2008/01/24. doi: 10.1111/j.1600-0897.2007.00553.x 18211535.

57. Roan NR, Starnbach MN. Immune-mediated control of Chlamydia infection. Cell Microbiol. 2008;10(1):9–19. doi: 10.1111/j.1462-5822.2007.01069.x 17979983.

58. Poston TB, Darville T. Chlamydia trachomatis: Protective Adaptive Responses and Prospects for a Vaccine. Curr Top Microbiol Immunol. 2018;412:217–37. Epub 2016/04/02. doi: 10.1007/82_2016_6 27033698.

59. Murthy AK, Li W, Chaganty BK, Kamalakaran S, Guentzel MN, Seshu J, et al. Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection. Infect Immun. 2011;79(7):2928–35. Epub 2011/05/04. doi: 10.1128/IAI.05022-11 21536799; PubMed Central PMCID: PMC3191981.

60. Zhong G. Chlamydia Spreading from the Genital Tract to the Gastrointestinal Tract—A Two-Hit Hypothesis. Trends Microbiol. 2018;26(7):611–23. Epub 2018/01/01. doi: 10.1016/j.tim.2017.12.002 29289422; PubMed Central PMCID: PMC6003826.

61. Batteiger BE, Newhall WJt, Jones RB. Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis. Infect Immun. 1985;50(2):488–94. Epub 1985/11/01. 4055030; PubMed Central PMCID: PMC261980.

62. Peeling R, Maclean IW, Brunham RC. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect Immun. 1984;46(2):484–8. Epub 1984/11/01. 6209221; PubMed Central PMCID: PMC261559.

63. Yeruva L, Spencer N, Bowlin AK, Wang Y, Rank RG. Chlamydial infection of the gastrointestinal tract: a reservoir for persistent infection. Pathog Dis. 2013;68(3):88–95. Epub 2013/07/12. doi: 10.1111/2049-632X.12052 23843274; PubMed Central PMCID: PMC3751173.

64. Howe SE, Konjufca VH. Protein-coated nanoparticles are internalized by the epithelial cells of the female reproductive tract and induce systemic and mucosal immune responses. PloS one. 2014;9(12):e114601. Epub 2014/12/10. doi: 10.1371/journal.pone.0114601 25490456; PubMed Central PMCID: PMC4260873.

65. Howe SE, Konjufca VH. Per-oral immunization with antigen-conjugated nanoparticles followed by sub-cutaneous boosting immunization induces long-lasting mucosal and systemic antibody responses in mice. PloS one. 2015;10(2):e0118067. Epub 2015/02/25. doi: 10.1371/journal.pone.0118067 25710518; PubMed Central PMCID: PMC4339372.

66. Tifrea DF, Ralli-Jain P, Pal S, de la Maza LM. Vaccination with the recombinant major outer membrane protein elicits antibodies to the constant domains and induces cross-serovar protection against intranasal challenge with Chlamydia trachomatis. Infect Immun. 2013;81(5):1741–50. Epub 2013/03/13. doi: 10.1128/IAI.00734-12 [pii]. 23478318; PubMed Central PMCID: PMC3648024.

67. Howe SE, Sowa G, Konjufca V. Systemic and Mucosal Antibody Responses to Soluble and Nanoparticle-Conjugated Antigens Administered Intranasally. Antibodies. 2016;5(4). Artn 20 doi: 10.3390/Antib5040020 ISI:000387944000001. 31558001

68. Rosche KL, Aljasham AT, Kipfer JN, Piatkowski BT, Konjufca V. Infection with Salmonella enterica Serovar Typhimurium Leads to Increased Proportions of F4/80+ Red Pulp Macrophages and Decreased Proportions of B and T Lymphocytes in the Spleen. PloS one. 2015;10(6):e0130092. Epub 2015/06/13. doi: 10.1371/journal.pone.0130092 26068006; PubMed Central PMCID: PMC4466801.