AAEP FOCUS ON THE FIRST YEAR OF LIFE PROCEEDINGS / 2014

Review of Immune Function and Sepsis in the Neonatal Foal Kelsey A. Hart, DVM, PhD, DACVIM (LAIM)



are not specific for a particular pathogen; and (4) the complement system. The innate immune response is activated when highly conserved microbial antigens (aka pathogen associated molecular patterns, or PAMPs) are recognized by host immune cells (usually via binding to host pattern recognition receptors or PRRs). The goals of the innate immune response are two-fold – to eliminate invading extracellular and infected cells, and to activate the more specific and extensive adaptive immune response.

Take Home Message: Both the innate and adaptive arms of the immune system are present and competent in the neonatal foal, but specific responses are immature during the first weeks to months of life. Foals are dependent on colostral antibody transfer for appropriate immunoglobulin levels in the neonatal period, until their own endogenous immunoglobulin production is stimulated by antigen exposure during the first weeks and months of life. In addition, foal leukocytes exhibit some limitations in phagocytic and microbicidal capacity when compared to adult cells. Finally, foals exhibit some alterations in immunomodulatory cytokine production in comparison to adults, most consistently with decreased interferon-γ (IFN-γ) production. Together, these immature immune responses render the foal at increased risk for bacterial infection in the neonatal period.

The adaptive immune response is initially slower than the innate response, but is capable of efficiently and specifically targeting a much more diverse group of microbes. The main effector cells of the adaptive immune system are lymphocytes, which function in the two types of adaptive immunity: humoral and cell-mediated immunity. Two types of lymphocytes, B lymphocytes and T lymphocytes, work separately and together to combat the invading microbe through several mechanisms. B lymphocytes are predominantly activated by extracellular microbes, and respond by secreting antibodies that can neutralize infecting microbes and target them for destruction by other immune cells.

Author’s address—Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602; e-mail: [email protected].

I. A BRIEF REVIEW OF THE IMMUNE SYSTEM

T

he primary physiologic function of the immune system is to protect the host against microbial infection. Because this function is so vital to the host’s survival, the immune system employs a number of inter-related and sometimes redundant mechanisms to accomplish this aim (Fig. 1).

T lymphocytes are divided into helper T lymphocytes (CD4+) and cytotoxic T lymphocytes (CD8+). All T cells are activated by the presentation of processed foreign antigen via cell-surface receptors called Class I and II MHC molecules. Any cell infected with an intracellular pathogen can use Class I MHC pathways to present pieces of foreign antigen on the cell surface. Class I MHC + antigen then stimulates the activation and replication of CD8+ T cells specific for that antigen, ultimately resulting in destruction of infected cells. Professional antigen presenting cells (APCs) such as macrophages, dendritic cells, and B lymphocytes can also use Class II MHC pathways to present pieces of foreign antigen taken up from the external environment (i.e. after phagocytosis of extracellular microbes or microbial components). Antigen specific CD4+ T cells respond to antigen presented on Class II MHC molecules by proliferation and by secretion of chemical messengers called cytokines. These cytokines are vital for: (1) activation of B lymphocytes and CD8+ T cells specific for that antigen to facilitate clearance of the infection; (2) activation of macro-

The immune system can be divided into two main arms that play distinct but complementary roles at different times during the immune response: (1) innate immunity and (2) adaptive immunity. The innate immune system is responsible for the initial immune response to an invading microbe, and the key components of it are in place and active prior to the microbial invasion. The innate immune response is early and rapid, but is limited by restricted diversity and inefficient mechanisms to eliminate large microbial loads. Key components of the innate immune system include: (1) physical barriers to microbial invasion, including skin, mucosal epithelial cells, and microbial chemicals such as salivary enzymes; (2) circulating and tissue phagocytes (neutrophils, macrophages); (3) natural killer cells (cytotoxic lymphocytes that recognize and kill infected cells but

37

AAEP FOCUS ON THE FIRST YEAR OF LIFE PROCEEDINGS / 2014

phages to more efficiently phagocytose and kill the invading microbe; and (3) for stimulation and perpetuation of the inflammatory response that helps contain and combat microbial infection.

by recently activated CD4+ T cells and activated mast cells. Once differentiated into the TH2 phenotype, TH2 cells then produce large quantities of IL-4 as well as interleukin-5 (IL-5) and interleukin-13 (IL-13). These cytokines are important for stimulating the production of IgE and neutralizing IgG antibodies from B lymphocytes, as well as for activation of eosinophils, and alternate macrophage activation that promotes granuloma formation rather than enhanced microbial killing as with TH1 activation above. This type of immune response is most important for the elimination of parasitic and fungal infections, and may play a role in some allergies and immunemediated diseases as well.

Through these complementary mechanisms, the immune system is capable of responding to and eliminating a variety of different types of infectious microbes, from viruses to extracellular and intracellular bacteria to fungi and parasites. One key way in which the immune system tailors aspects of the immune response to different types of invading organisms is through differentiation of helper T lymphocytes (CD4+) into specific subsets of cells with different effector functions. The best defined subsets of activated CD4+ cells are the T H1 and TH2 subsets, distinguished by the cytokines they produce and thus by the aspects of the immune response these cytokines stimulate. CD4+ cells are encouraged to differentiate into T H1 cells by the presence of interferon gamma (IFN-γ) and interleukin-12 (IL-12) from activated innate immune and inflammatory cells such as macrophages, dendritic cells, and NK cells. TH1 cells then secrete large amounts of IFN-γ, which subsequently further activates of macrophages and neutrophils to enhance microbial clearance, and stimulates B lymphocytes to produce antibodies important for targeting extracellular microbes for phagocytosis (opsonizing antibodies). The T H1 response, then, is critical for directing the immune response towards efficient clearance of intracellular, phagocytosed microbes.

One other important component of the adaptive immune response is the development of immunologic memory, which enables the immune system to “remember” microbes it has previously encountered and to respond more swiftly and aggressively upon repeat infection with the same organism. Immunologic memory is produced by the maintenance of small numbers of antigen-specific circulating B and T lymphocytes that are able to be fully and rapidly activated when they encounter the previous invader.

II. IMMUNE FUNCTION IN THE NEONATAL FOAL The neonatal foal is essentially immunocompetent but immunologically naïve at birth. Thus, the neonatal foal is capable of mounting an immune response to microbial infection, but due to the relative isolation from antigenic

In contrast, a TH2 response develops in the absence of IFN-γ and IL-12 and in the presence of interleukin-4 (IL-4), produced 22

AAEP FOCUS ON THE FIRST YEAR OF LIFE PROCEEDINGS / 2014 stimulation while in utero, the foal’s immune system is not fully “up and running” until antigen exposure occurs after parturition. The newborn foal’s immune system contains all the key cellular players for both innate and adaptive immunity – functional phagocytes, antigen presenting cells, and B and T lymphocytes – but these players have yet to be “fully trained” and thus are not always present in comparable numbers or operating at full capacity as compared to adult horses. In addition, at birth, circulating levels of key soluble immune factors such as immunoglobulins, complement, lactoferrin and some cytokines are also significantly lower than in adults.

by decreased immunoglobulin and complement levels, some studies have documented decreased phagocytic capacity in foal neutrophils even when exposed to adult serum for opsonization.8 Recently in our laboratory, we found that foal neutrophils adequately phagocytosed both gram negative and gram positive organisms without the need for opsonization with equine serum (either autologous or adult serum), but that the degree of phagocytosis was lower in foals than adult horses and was somewhat pathogen specific (Hart, 2013, unpublished data). Thus, neonatal foal neutrophils may exhibit some limitations in phagocytic capacity that may be related to certain pathogens or to the amount of pathogen present.

Repeated antigenic stimulation during the early postnatal period is vital for getting the foal’s immune system into gear. The specific time at which all aspects of the foal’s immune response develop to a point comparable to adult horses is not known, but evidence suggests this occurs sometime during the first year of life and probably varies for different components of the immune response.1 Until that time, the foal seems to be more vulnerable to pathogenic and opportunistic infections than adult horses. 1 Similar immunologic immaturity occurs in neonates of many species, and is partially compensated by passive transfer of immunity from the mother to the fetus or neonate. Equine placentation does not permit placental transfer of immune factors, so the neonatal foal is essentially dependent on factors present in colostrums for transfer of passive immunity from the dam. Our understanding of specific factors contributing to immunologic immaturity in the neonatal foal is at present incomplete, though active research in this area is ongoing. Specific differences in immune function between neonatal foals and adult horses are detailed below.

Neutrophil oxidative burst activity is important for the destruction of phagocytosed and free microbes, and also plays a key role in the innate immune and inflammatory response. While several studies have documented that neutrophil oxidative burst activity is comparable to adult activity in foals < 1 week of age,7,8 other studies have shown that the leukocyte killing capacity in blood from 2 day-old foals is significantly less than that of adult horses, and remains lower than adult horse levels until 3 months of age.6 In our laboratory, we have recently documented that neutrophil production of reactive oxygen species in response to a variety of stimulants is present in foals age 2-8 days but is in general less than is observed in mature horses (Hart, 2013, unpublished data). When considered in concert, the evidence for decreased opsonic capacity in foal serum, potentially limited phagocytic capacity for specific pathogens in foal neutrophils, and potentially impaired oxidative burst and killing activity in foal neutrophils, suggests that the innate immune response in the foal may be limited in some aspects and may play a role in susceptibility of foals to bacterial infection.

Innate Immunity in the Neonatal Foal

Adaptive Immunity in the Neonatal Foal

As described above, the innate immune response provides the first line of defense against microbial infection, and is critical for alerting the immune system about any infectious threats and initiating the more specific and fine-tuned adaptive immune response. Key players in the innate immune response include soluble factors like immunoglobulins, complement, and lactoferrin, and phagocytic cells like neutrophils, macrophages and dendritic cells. Prior to ingestion of colostrum, neonatal foals are hypogammaglobulinemic and have significantly lower levels of lactoferrin in comparison with foals after colostral ingestion and adult horses.2,3 In addition, complement activity in neonatal foals has been shown to be significantly decreased in comparison to adult horses (at just 13% of adult activity at birth).4

In addition, the adaptive immune response does not appear to be fully efficient and specific in the neonatal foal as in the mature horse. Antigen presenting cells (APCs) function as the vital bridge between the innate and adaptive immune systems. Exposure to and uptake of specific antigen by an immature APC, as well as a specific cytokine milieu, leads to maturation of the APC, enabling it to more efficiently present antigen via up-regulated MHC Class II. Mature APCs produce specific cytokines such as IFN-γ that stimulate and direct the adaptive immune response. Several studies have documented decreased or immature antigen presenting capacity in foals, as evidenced by decreased MHC Class II expression on lymphocytes, macrophages and dendritic cells7,9 and decreased numbers of mature dendritic cells in young foals as compared to adult horses.10

Phagocytes counts in newborn foals are comparable to adult horses, but these cells do not appear to function as robustly and efficiently at microbial uptake in the foal as the adult horse. Some studies have suggested that this decreased phagocytic capacity in foal neutrophils is due to decreased opsonic capacity of foal serum, as phagocytic function improved to adult levels when bacteria or yeast organisms were pre-opsonized with adult horse serum.5-7 While reduced opsonic capacity in foal serum (especially with pre-colostral serum) is well-supported

Impaired APC signaling may be vital in explaining other limitations in adaptive immune responses documented in neonatal foals. Despite comparable or even increased numbers of lymphocytes in neonatal foals as compared to adult horses,7,11 lymphocyte subpopulation distribution and lymphocyte activation and proliferation differ in neonatal foals. While circulating B lymphocyte counts are significantly higher in foals from birth to 6 months of age than in mature horses,12 23

AAEP FOCUS ON THE FIRST YEAR OF LIFE PROCEEDINGS / 2014 and foal B cells at birth are developmentally competent to produce a variety of immunoglobulin isotypes,3 some immunoglobulin isotype concentrations in foals remain significantly lower than in adult horses for during the first year of life. The presence of colostrally-derived maternal antibodies in the weeks following parturition may limit the foal’s endogenous production of immunoglobulins during this period to some degree. However, B cell numbers increase steadily during the first months of life, concurrently with an increase in foal IgM and IgG production, demonstrating activation of the humoral immune system with antigen exposure.12 Despite this, foal B lymphocytes still exhibit decreased production of IgG(b) throughout the first year of life.1

inadequate colostral intake or poor quality colostrum dramatically increases the neonatal foal’s risk of infection. The contribution of colostral immunoglobulins to the foal’s humoral immunity is well known, but other colostral factors – such as lactoferrin, inflammatory cytokines, and even functional leukocytes – also play a key role in the foals’ immune response during the neonatal period. Thus, ensuring adequate transfer of passive immunity with prompt colostrum ingestion is a critical first step to immunologic support for the neonatal foal. However, even foals with adequate transfer of passive immunity have an increased risk of systemic bacterial infection (e.g. sepsis) during the neonatal period as compared with adult horses. Some of the partial deficiencies in the foals’ naive innate and adaptive immune responses discussed above – such as reduced plasma opsonin capacity, inefficient phagocytic and oxidative burst activity, immature antigen presenting capacity, and immature B and T lymphocyte function – likely play a role in this increased risk, as the foal’s immune system may be less effective and efficient at clearing infectious microbes, which could permit spread of a localized infection. In addition, impaired cortisol responses during the neonatal period in foals22-24 could permit an overwhelming systemic inflammatory response to develop more easily in foals than adults, perpetuating the development of the systemic inflammatory response syndrome and septic shock in neonatal foals.

CD4+ and CD8+ T cells also increase steadily in numbers during the first few months of life in foals. 12 However, foal T lymphocytes exhibit decreased proliferative response to several mitogens, in particular concavalin A, at birth, though this quickly increases to adult levels. The decreased Class II MHC expression noted on foal T cells at birth also gradually increases to adult levels by 4 months of age, suggesting progressive development of memory T lymphocytes.7 Considered together, these findings in regards to B and T cell number and function during the neonatal period in the foal suggest that these cells are in general functional at birth but require the antigenic stimulation that occurs during early life in order to develop their functional efficiency to adult capacity.

However, this degree of immunologic immaturity fails to fully explain the foal’s propensity for development of opportunistic infections rarely seen in adult horses (e.g. Rhodococcus equi, Pneumocystis carinii, and systemic candidiasis), which may suggest an integrated immune or immunoendocrine deficiency in the neonatal foal that is not elucidated by evaluation of specific cell numbers or function.

Finally, several studies have examined the effect of age and illness on cytokine gene expression in foal peripheral blood mononuclear cells (PBMCs) and neutrophils. While basal expression of many cytokines (IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p35 and TNF-α) in PBMCs did not change in foals during the first month of life, expression of IFN-γ was low at birth and increased significantly over the first month of life.13-15 Both basal and pathogen-stimulated expression of IFN-γ in PBMCs were lower in foals at birth and increased with age. 16 In addition, evaluation of cytokine profiles in sick and septic foals showed these foals failed to increase PBMC IFN-γ expression despite the presence of bacterial infection.15,17,18 Recent evidence suggests that age-associated changes in methylation of the IFN- γ gene promoter appear to play a role in limited IFNγ production capacity in newborn foals.19 In concert, these findings suggest that the neonatal foal’s ability to mount an efficient TH1 response may be somewhat impaired. However, experimental infection with a low inoculum of Rhodococcus equi in foals and treatment of 1-2 week old foals with an immunomodulator both results in increased IFN-γ production,20,21 suggesting that foal leukocytes might be capable, with the proper stimulus, of producing an acceptable TH1-type response.

As discussed above, a TH1 immune response is vital for appropriate immunity to bacterial invaders, and is initiated by cells of the innate immune system and APCs that produce IFNγ and IL-12 after recognition of infecting bacteria. IFN-γ and IL-12 induce naïve CD4+ T cells towards a T H1 type phenotype, which then produce additional IFN-γ, TNF-α, and lymphotoxin (LT). IFN-γ stimulates macrophage activation, leading to enhanced microbial killing, and induces the production of complement-binding and opsonizing antibodies (IgG isotypes) by B lymphocytes, all of which are vital for clearance of bacterial infection, particularly with intracellular organisms. Thus, an age-related limitation in IFN-γ production may indicate a TH1 deficiency in the neonatal foal that could significantly impact the foal’s ability to effectively clear some bacterial pathogens, predisposing the neonatal foal to infection with specific intracellular pathogens like R. equi and P. carinii. The above evidence suggests that a relative deficiency in antigen presenting capacity due to APC immaturity (as detailed above) may play a role in IFN-γ deficiency in the neonatal foal that could be related to the pathogenesis of such infections in the foal.

III. CONSEQUENCES AND MANAGEMENT OF “IMMUNOLOGIC IMMATURITY” IN THE FOAL Since the foal depends so heavily on colostral transfer of passive immunity for immune support during the first few weeks of life, failure of this transfer of passive immunity due to 24

AAEP FOCUS ON THE FIRST YEAR OF LIFE PROCEEDINGS / 2014

REFERENCES 1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Giguere S, Polkes A. Immunologic disorders in neonatal foals. Vet Clin Equine 2005;21:241-272. Barton M, Hurley D, Norton N, et al. Serum immunoglobulin G and lactoferrin concentrations in healthy and septicemic neonatal foals. J Vet Intern Med 2004;18:413. Tallmadge R, McLaughlin K, Secor E, et al. Expression of essential B cell genes and immunoglobulin isotypes suggests active development and gene recombination during equine gestation. Dev Comp Immunol 2009;33:1027-1038. Bernoco M, Liu I, Willits N. Hemolytic complement activity and concentrations of its third component during maturation of the immune response in colostrum-deprived foals. Am J Vet Res 1994;55:928-933. Grondahl G, Sternberg S, Jensen-Waern M, et al. Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli. Equine Vet J 2001;33:670-675. Demmers S, Johannisson A, Grondahl G, et al. Neutrophil function and serum IgG in growing foals. Equine Vet J 2001;33:676-680. Flaminio M, Rush B, Davis E, et al. Characterization of peripheral blood and pulmonary leukocyte function in healthy foals. Vet Immunol Immunopathol 2000;73:267285. McTaggart C, Yovich J, Penhale J, et al. A comparison of foal and adult horse neutrophil function using flow cytometric techniques. Res Vet Sci 2001;71:73-79. Flaminio M, Borges A, Nydam D, et al. The effect of CpGODN on antigen presenting cells of the foal. J Immune Based Ther Vaccines 2007;25:1. Merant C, Breathnach C, Kohler K, et al. Young foal and adult horse monocyte-derived dendritic cells differ by their degree of phenotypic maturity. Vet Immunol Immunopathol 2009;131:1-8. Smith R, Chaffin M, Cohen N, et al. Age-related changes in lymphocyte subsets of Quarter Horse foals. Am J Vet Res 2002;63:531-537. Flaminio M, Rush B, Shuman W. Peripheral blood lymphocyte subpopulations and immunoglobulin concenrations in healthy foals and foals with Rhodococcus equi pneumonia. J Vet Intern Med 1999;13:206-212. Boyd N, Cohen N, Lim W-S, et al. Temporal changes in cytokine expression in foals during the first month of life. Vet Immun Immunopath 2003;92:73-85. Ryan C, Giguere S, Hagen J, et al. Effect of age and mitogen on the frequency of interleukin-4 and interferon gamma secreting cells in foals and adult horses as assessed by an equine-specific ELISPOT assay. Vet Immunol Immunopathol 2010;133:66-71. Castagnetti C, Mariella J, Pirrone A, et al. Expression of interleukin-1beta, interleukin-8, and interferon gamma in blood samples obtained from healthy and sick neonatal foals. Am J Vet Res 2012;73:1418-1427. Liu T, Nerren J, Liu M, et al. Basal and stimulus-induced cytokine expression is selectively impaired in peripheral

18.

19.

20.

21.

22.

23.

24.

25

blood mononuclear cells of newborn foals. Vaccine 2009;27:674-683. Gold J, Perkins G, Erb H, et al. Cytokine profiles of peripheral blood mononuclear cells isolated from septic and healthy neonatal foals. J Vet Intern Med 2007;21:482488. Pusterla N, Magdesian K, Mapes S, et al. Expression of molecular markers in blood of neonatal foals with sepsis. Am J Vet Res 2006;67:1045-1049. Sun L, Gong Z, Oberst E, et al. The promoter region of interferon-gamma is hypermethylated in neonatal foals and its demethylation is associated with increased gene expression. Dev Comp Immunol 2013;39:273-278. Sturgill T, Giguere S, Franklin R, et al. Effects of inactivated parapoxvirus ovis on the cumulative incidence of pneumonia and cytokine secretion in foals on a farm with endemic infections caused by Rhodococcus equi. Vet Immunol Immunopathol 2011;140:237-243. Jacks S, Giguere S, Crawford P, et al. Experimental infection of neonatal foals with Rhodococcus equi triggers adult-like gamma interferon induction. Clin Vaccine Immunol 2007;14. Hart K, Barton M, Vandenplas M, et al. Effects of lowdose hydrocortisone therapy on immune function in neonatal horses. Pediatr Res 2011;70:72-77. Hart K, Heusner G, Norton N, et al. Hypothalamicpituitary-adrenal axis assessment in healthy term neonatal foals utilizing a paired low dose/high dose ACTH stimulation test. J Vet Intern Med 2009;23:344-351. Hart K, Slovis N, Barton M. Hypothalamic-pituitaryadrenal axis dysfunction in hospitalized neonatal foals. J Vet Intern Med 2009;23:901-912.