Being TB a long-lasting affliction, most research on the immune responses to TB has addressed the chronic stages. In contrast, the earliest contacts of bacilli with cells responsible of the first defenses have been rather neglected, until recent years. Neutrophils (PMNs) are short-lived leukocytes, functioning as the primary defense against microorganisms [6, 7], with special ability to kill pathogens by phagocytosis, degranulation and the formation of extracellular traps [8-11]. The activation of neutrophils is associated with changes in the spatial and temporal expression of certain molecules [12, 13] such as CD16b, CD11b and CD66b, molecules responsible for adhesion, degranulation and migration, and are therefore well established as neutrophil activation markers [13-19]. On the other hand, it is documented that intracellular pathogens causing chronic infections can co-opt the pathway of a pair of molecules, PD-1 and its ligand PD-L1, involved in decreasing crucial T cell responses, provoking a phenomenon called "T cell exhaustion" . In physiological conditions, this pathway is important for peripheral tolerance and the control of adaptive immune responses , but it has been barely explored in TB.
In this work we wanted to assess the phenotype of neutrophils during the very early interactions with three different strains from the
2. Materials and methods
This research was performed on healthy competent volunteers in accordance with the Declaration of Helsinki of the world Medical Association, and the Mexican General Health Law regarding research. The ethics committee of the National School of Biological Sciences ENCB-IPN approved this study (permission number: “Protocolo #CEI-ENCB 011/2013”) and informed written consent was obtained from donors.
2.2. Bacterial strains and cultures
2.3. Isolation of human neutrophils
Human blood neutrophils were isolated from healthy donors using two gradients: Histopaque 1119 (Sigma-Aldrich, St. Louis, MO, USA. Cat. 11191) for 20 min at 800g and Percoll (GE Healthcare Bio-sciences AB, Uppsala, Sweden. Cat. 17-0891-01) at the densities indicated next: 1105 g/mL (85%), 1100 g/mL (80%), 1093 g/mL (75%), 1087 g/mL (70%) y 1081 g/mL (65%), for 20 min at 800g.
2.4. Stimulation and staining of neutrophils
106 healthy neutrophils/mL of RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) were used either unstimulated or stimulated with 10 nM Phorbol 12-Myristate 13-Acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA. Cat. P-81-39), 5 μg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA. Cat. L-3755),
Statistics were performed with Two-way ANOVA using Bonferroni
3.1. Neutrophils purity
Blood neutrophils from healthy donors were enriched to about 90% purity, which was checked through staining with Hematoxylin and Eosin (H&E) as well as by flow cytometry parameters such as cell size (FSC) vs. granularity/internal complexity (SSC) (Figure 1 A, B). PMNs were incubated either with culture medium alone, LPS, PMA or the various mycobacteria at two different multiplicity of infection (MOI: 0.1, 1) of
Mycobacterium affects neutrophils integrity at early times of interactions
By means of flow cytometry we analyzed the neutrophils incubated either with culture medium alone, LPS, PMA,
3.3. Mycobacteria do not activate neutrophils at early times of interaction
Blood neutrophils incubated separately with three different strains of Mycobacteria (BCG, Canetti and Mtb) were evaluated for the expression of CD16b (Figure 3), CD11b and CD66b (Figure 4), at 15, 30 and 60 min incubation. Although these molecules are constitutively expressed on neutrophils, it is well documented that neutrophils modify the expression of these markers upon activation [13, 15, 17, 19, 28-30].
Neutrophils incubated with mycobacteria did not display changes in the percentages of expression of CD16b (Figure 3A), CD11b, CD66b (Figure 4A, C) at the 3 time-points evaluated. There was, however, a decreased in the
3.4. Mycobacteria rapidly induce pro-inhibitory molecules on neutrophils
Mycobacteria have evolved diverse mechanisms to escape, divert or even subvert the immune responses [4, 31]. The molecular pair PD1-PD-L1 has been recently shown to induce a phenomenon called T cell exhaustion, and the expression of these two molecules has been shown manipulated by certain pathogens for their advantage [32, 33]. The percentage of PD1 expression in neutrophils incubated with mycobacteria increased only with Mtb H37Rv (figure 5A, black arrows) since 15 min, compared with basal expression levels. In contrast, PMNs interacting with the other mycobacterial strains did not modify the percentage of PD-1 expression (Figure 5A). Regarding MFI we observed a decrease in PD-1 intensity in neutrophils incubated with Mtb H37Rv MOI:1 at 15 and 30 min, but an increase at 60 min (figure 5B, black arrow). With respect to PD-1 Ligand (PD-L1), the percentage increased in neutrophils incubated with BCG and
We aimed at assessing activation as well as pro-inhibitory molecules on neutrophils from healthy donors using flow cytometry, thus PMNs were incubated for short periods with three different strains of mycobacteria. As positive controls for PMN activation at the time points evaluated, we used two standard components, a microbial one such as lipopolysaccharide (LPS), and a chemical one such as phorbol myristate acetate (PMA). At least two main FACS criteria were evaluated, the percentage of cells and the median fluorescence intensity (MFI). The latter directly reflects the intensity of expression of a given molecule in the cells evaluated.
In flow cytometry the emission of autofluorescence in both animal and plants cells has been indicative of cell damage or even cell death. In addition, the reduction of NADH induces intracellular fluorescence, also indicating cell damage [22-27]. When we cultured mycobacteria with neutrophils, these increased their autofluorescence emission since 15 min, compared with neutrophils in medium alone. Previously, Perskvist
Few and recent studies have focused on the effects that mycobacteria might have on neutrophils [40-42], for instance whether mycobacteria can induce neutrophil extracellular traps, as seen when PMNs interact with other microorganisms . Of note, even the phenotype of neutrophils during and after their interaction with mycobacteria remains poorly addressed.
From the set of molecules that we measured, some (CD16b, CD11b, CD66b) are well established neutrophil activation markers, while the others (PD1/PD1-L) are very important pro-inhibitory molecules. Interestingly, however, even at the longest time of incubation with mycobacteria (60 min), we did not observe statistically significant differences in the percentages of PMNs expressing CD16b, CD11b or CD66b, compared to neutrophils incubated with the control substances LPS, PMA or with culture medium alone. In contrast, it is known that certain components from microorganisms such as
On the other hand, decrease in surface CD16b in neutrophils is also related to the activation of these cells [29, 30]. When we analyzed the median fluorescence intensity (MFI) of CD16b on mycobacteria-incubated neutrophils, we did not find significant differences compared to the controls, although there was a slight decrease at 15 minutes in cells incubated with Mtb H37Rv compared to freshly isolated neutrophils. Regarding the expression of CD11b and CD66b, both LPS and PMA (used as positive controls for stimulation), increased the expression of these two markers since 15 minutes compared to neutrophils in medium alone, indicative of the intact capacity of neutrophils to respond. Although there was a subtle decrease in CD66b expression at 15 minutes of incubation with bacilli, compared to freshly isolated neutrophils.
Previous studies have evaluated the expression of CD16b and CD66b as activation markers in neutrophils from patients with active TB . Likewise, in healthy neutrophils incubated with clinical isolates of Mtb, the expression of CD16, CD69 and CXCR2 was evaluated, but only at 3 and 18 h . Hilda
The PD-1/PD-L1 pathway is important for the establishment and maintenance of peripheral tolerance as well as to modulate immune and inflammatory responses to pathogens [21, 48]. This pathway has been found altered during chronic viral [49, 50], parasitic , fungal  and bacterial  infections, provoking a phenomenon known as “exhaustion” in CD8+T cells, exerting an inhibitory effect related to pathogens evasion of immune responses . Apparently, Mtb H37Rv can also modulate and manipulate the PD/PD-L1 path not only in T cells, but also in antigen presenting cells and even in elements of the innate immune response, such as NK cells . However, this pair of molecules has been poorly explored in neutrophils, especially in the interplay with microorganisms. Therefore, we sought to evaluate also the neutrophils expression of PD-1 and PD-L1 in the early response to mycobacteria. There was an apparent increase in the proportion of neutrophils expressing surface PD-1 following incubation with Mtb H37Rv (MOI 1), which was more evident at 60 minutes; although not statistically significant. These results are interesting and opposed to data by Yao
Our results indicate that, while neutrophils are readily stimulated upon short time of incubation with either LPS or PMA, they do not get activated during early interactions with three mycobacteria of different virulence. This is revealed by the PMNs inability to modify activation molecules which are involved in crucial processes such as migration, adhesion, phagocytosis and degranulation. However, at these early times of interactions with mycobacteria, there are indeed alterations occurring in other important molecules in PMNs, such as PD1/PD1-L.
It is conceivable that early during a mycobacterial infection neutrophils might encounter and interact with other recruited cells of the immune system while still expressing inhibitory molecules such as PD-1 or PD-L1, thus limiting the intensity of the response at the site of infection. The fact that neutrophils infected with Mtb express both the receptor and the ligand of PD-1 could be related to an autocrine regulation of this population. Further experiments are required to understand the role of the early innate immune response against
List of abbreviations
I Estrada-García, S Estrada-Parra, R Chacon-Salinas, J Calderon-Amador and L Flores-Romo are members of the National System of Researchers (SNI) from Mexico. I Estrada-García (Conacyt project 221002), S Estrada-Parra, R Chacon-Salinas are fellow-holders from EDI and COFAA. M Orozco-Uribe, J Castañeda-Casimiro and L Donis-Maturano were fellow-holders from the National Council for Science and Technology, Conacyt. LDM was a postdoctoral fellow from Conacyt. Authors acknowledge the help from V Rosales at the FACS facilities.
Maartens, G. and R.J. Wilkinson, Tuberculosis.Lancet, 2007. 370(9604): p. 2030-43.
(WHO), W.H.O., Global Tuberculosis Report 2013.2013.
Martineau, A.R., et al., Neutrophil-mediated innate immune resistance to mycobacteria.J Clin Invest, 2007. 117(7): p. 1988-94.
Gupta, A., et al., Mycobacterium tuberculosis: immune evasion, latency and reactivation.Immunobiology, 2012. 217(3): p. 363-74.
Zuniga, J., et al., Cellular and humoral mechanisms involved in the control of tuberculosis.Clin Dev Immunol, 2012. 2012: p. 193923.
Zychlinsky, A., Y. Weinrauch, and J. Weiss, Introduction: Forum in immunology on neutrophils.Microbes Infect, 2003. 5(14): p. 1289-91.
Pruchniak, M.P., M. Arazna, and U. Demkow, Life of neutrophil: From stem cell to neutrophil extracellular trap.Respir Physiol Neurobiol, 2013. 187(1): p. 68-73.
Brinkmann, V., et al., Neutrophil extracellular traps kill bacteria.Science, 2004. 303(5663): p. 1532-5.
Simon, H.U., Neutrophil apoptosis pathways and their modifications in inflammation.Immunol Rev, 2003. 193: p. 101-10.
Zakhireh, B., L.H. Block, and R.K. Root, Neutrophil function and host resistance.Infection, 1979. 7(2): p. 88-98.
Fox, S., et al., Neutrophil apoptosis: relevance to the innate immune response and inflammatory disease.J Innate Immun, 2010. 2(3): p. 216-27.
Smith, J.A., Neutrophils, host defense, and inflammation: a double-edged sword.J Leukoc Biol, 1994. 56(6): p. 672-86.
Nair, K.S. and S.M. Zingde, Adhesion of neutrophils to fibronectin: role of the cd66 antigens.Cell Immunol, 2001. 208(2): p. 96-106.
Witko-Sarsat, V., et al., Neutrophils: molecules, functions and pathophysiological aspects.Lab Invest, 2000. 80(5): p. 617-53.
David, A., et al., Interaction of proteinase 3 with CD11b/CD18 (beta2 integrin) on the cell membrane of human neutrophils.J Leukoc Biol, 2003. 74(4): p. 551-7.
Schroder, A.K., et al., Crosslinking of CD66B on peripheral blood neutrophils mediates the release of interleukin-8 from intracellular storage.Hum Immunol, 2006. 67(9): p. 676-82.
Feuk-Lagerstedt, E., et al., Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.J Immunol, 1999. 163(10): p. 5592-8.
Skubitz, K.M., K.D. Campbell, and A.P. Skubitz, CD66a, CD66b, CD66c, and CD66d each independently stimulate neutrophils.J Leukoc Biol, 1996. 60(1): p. 106-17.
Schmidt, T., et al., CD66b overexpression and homotypic aggregation of human peripheral blood neutrophils after activation by a gram-positive stimulus.J Leukoc Biol, 2012. 91(5): p. 791-802.
Hofmeyer, K.A., H. Jeon, and X. Zang, The PD-1/PD-L1 (B7-H1) pathway in chronic infection-induced cytotoxic T lymphocyte exhaustion.J Biomed Biotechnol, 2011. 2011: p. 451694.
Folkl, A. and D. Bienzle, Structure and function of programmed death (PD) molecules.Vet Immunol Immunopathol, 2010. 134(1-2): p. 33-8.
Barenboim GM, D.A., Turoverov KK (Eds.), Luminescence of Biopolymers and Cells. 1969, New York: Plenum Press.
Chance, B. and B. Thorell, Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry.J Biol Chem, 1959. 234: p. 3044-50.
Davis, R.P. and M. Canessa-Fischer, Spectrofluorometric Identification of Reduced Pyridine Nucleotide in the Intact Isolated Urinary Bladder of the Toad.Anal Biochem, 1965. 10: p. 325-43.
Haitinger, M., Fluoresenz-Mikroskopie. 1959, Leipzig: Academie-Verlag.
Pearse, A., Histochemistry: Theoretical and Applied. 1972, London: churchill Livingstone Co.
Udenfriend, S., Fluorescence Assay in Biology and Medicine. 1969, New York: Academic Press.
Silveira, A.A., et al., Simvastatin abrogates inflamed neutrophil adhesive properties, in association with the inhibition of Mac-1 integrin expression and modulation of Rho kinase activity.Inflamm Res, 2013. 62(2): p. 127-32.
Leino, L. and E.M. Lilius, The up-and down-modulation of immunoglobulin G Fc receptors and complement receptors on activated human neutrophils depends on the nature of activator.J Leukoc Biol, 1992. 51(2): p. 157-63.
Nagarajan, S., N.H. Fifadara, and P. Selvaraj, Signal-specific activation and regulation of human neutrophil Fc gamma receptors.J Immunol, 2005. 174(9): p. 5423-32.
Meena, L.S. and Rajni, Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv.FEBS J, 2010. 277(11): p. 2416-27.
Jurado, J.O., et al., Programmed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis.J Immunol, 2008. 181(1): p. 116-25.
Donis-Maturano, L., Caracterización fenotípica de las subpoblaciones de linfocitos T en parénquima pulmonar murino durante la infección con Mycobacterium tuberculosis, in Departamento de Inmunología2006, Escuela Nacional de Ciencias Biológicas (ENCB)-IPN: México, D.F.
Perskvist, N., et al., Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway.J Immunol, 2002. 168(12): p. 6358-65.
Blomgran, R., et al., Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells.Cell Host Microbe, 2012. 11(1): p. 81-90.
Aga, E., et al., Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major.J Immunol, 2002. 169(2): p. 898-905.
Corleis, B., et al., Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.Cell Microbiol, 2012. 14(7): p. 1109-21.
Ramos-Kichik, V., et al., Neutrophil extracellular traps are induced by Mycobacterium tuberculosis.Tuberculosis (Edinb), 2009. 89(1): p. 29-37.
Urban, C.F., et al., Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.Cell Microbiol, 2006. 8(4): p. 668-76.
Pedrosa, J., et al., Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice.Infect Immun, 2000. 68(2): p. 577-83.
Kisich, K.O., et al., Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils.Infect Immun, 2002. 70(8): p. 4591-9.
Persson, A., et al., Induction of apoptosis in human neutrophils by Mycobacterium tuberculosis is dependent on mature bacterial lipoproteins.Microb Pathog, 2009. 47(3): p. 143-50.
Soler-Rodriguez, A.M., et al., Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14.J Immunol, 2000. 164(5): p. 2674-83.
Alvarez, M.E., et al., Neutrophil signaling pathways activated by bacterial DNA stimulation.J Immunol, 2006. 177(6): p. 4037-46.
Aleman, M., et al., Activation of peripheral blood neutrophils from patients with active advanced tuberculosis.Clin Immunol, 2001. 100(1): p. 87-95.
Pokkali, S., et al., Phenotypic modulation in Mycobacterium tuberculosis infected neutrophil during tuberculosis.Indian J Med Res, 2009. 130(2): p. 185-92.
Hilda, J.N., A. Selvaraj, and S.D. Das, Mycobacterium tuberculosis H37Rv is more effective compared to vaccine strains in modulating neutrophil functions: an in vitro study.FEMS Immunol Med Microbiol, 2012. 66(3): p. 372-81.
Odorizzi, P.M. and E.J. Wherry, Inhibitory receptors on lymphocytes: insights from infections.J Immunol, 2012. 188(7): p. 2957-65.
Barber, D.L., et al., Restoring function in exhausted CD8 T cells during chronic viral infection.Nature, 2006. 439(7077): p. 682-7.
Freeman, G.J., et al., Reinvigorating exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade.J Exp Med, 2006. 203(10): p. 2223-7.
Gutierrez, F.R., et al., Regulation of Trypanosoma cruzi-induced myocarditis by programmed death cell receptor 1.Infect Immun, 2011. 79(5): p. 1873-81.
Lazar-Molnar, E., et al., The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum.Proc Natl Acad Sci U S A, 2008. 105(7): p. 2658-63.
Alvarez, I.B., et al., Role played by the programmed death-1-programmed death ligand pathway during innate immunity against Mycobacterium tuberculosis.J Infect Dis, 2010. 202(4): p. 524-32.
Yao, S., et al., PD-1 on dendritic cells impedes innate immunity against bacterial infection.Blood, 2009. 113(23): p. 5811-8.
McNab, F.W., et al., Programmed death ligand 1 is over-expressed by neutrophils in the blood of patients with active tuberculosis. Eur J Immunol, 2011. 41(7): p. 1941-7.