Charla 1. Mecanismos de evasión de la respuesta inmune: ejemplos con Anisakis. XXI congreso de la SOCEPA. Pontevedra. Julio, 2019

1. Mecanismos de evasión de
la respuesta inmune:
ejemplos con Anisakis
Carmen Cuéllar
Parasitología
Facultad de Farmacia
UCM
SOCEPA, 2019

2. MECANISMOS DE EVASIÓN DE LA RESPUESTA INMUNE
TÁCTICAS DE EVITACIÓN
TÁCTICAS DE ENMASCARAMIENTO
TÁCTICAS DE OCULTAMIENTO
TÁCTICAS DE INMUNOMODULACIÓN

3. Cuéllar C, Perteguer MJ, De Las Heras B. Effects of Anisakis simplex on nitric oxide production in J774 macrophages. Scand J Infect Dis.
1998;30(6):603‐6.
Although the parasite cannot remain alive for a long time, their
products may contribute to the development of the chronic disease.

4. Cuéllar C, Perteguer MJ, Rodero M. Presence of IL‐4‐like molecules in larval excretory‐secretory products and crude extracts from Anisakis
simplex. Scand J Immunol. 2001 May;53(5):483‐8.
The parasite may modulate the Th1‐Th2 dichotomy for its own benefit by
mucosal inflammation control in an attempt to avoid the larval expelling.

5. Anisakis simplex: The activity of larval products on the
complement system
García‐Hernández P, Rodero M, Cuéllar C. Anisakis simplex: the activity of larval products on the complement system. Exp Parasitol. 2007
Jan;115(1):1‐8.
García‐Hernández P, Rodero M, Cuéllar C. Study of the effect of Anisakis simplex larval products on the early and late components in the classical
complement pathway. J Parasitol. 2009 Feb;95(1):240‐1.
García‐Hernández P, Rodero M, Gisbert‐Criado R, Puente P, Pelayo V, Andreu‐Ballester JC, Cuéllar C. The effect of anti‐Anisakis simplex antibody
levels on C3 and C4 complement components in human sera. J Helminthol. 2012 Jun;86(2):197‐201.
CE and ES products from A. simplex interact with C3 and C2 complement proteins.
The presence of an association between high levels of anti‐A. simplex antibodies and C3/C4 deficiency has been proved.
This modulatory activity could facilitate A. simplex L3 to migrate across host tissues as to survive in an atypical host.

6. LARVA
STOMACH SMALL INTESTINE
Ag+LARVA
Perteguer MJ, Rodero M, Flores JM, Dórea RC, Cuéllar C. Cellular immune responses in mice immunized with Anisakis simplex larval antigens.
Parasitol Res. 2001 May;87(5):396‐404.
Cellular immune responses in mice immunized with Anisakis
simplex larval antigens.
Histological analysis showed that the most prominent lesions were gastric and
intestinal in animals infected orally with one larva.

7. CD45+TCR‐
CD45+TCR +
CD11b+
CD4+CD8a+
CD4‐CD8a+
CD8a+TCR +
CD8a+TCR ‐
Perteguer MJ, Rodero M, Flores JM, Dórea RC, Cuéllar C. Cellular immune responses in mice immunized with Anisakis simplex larval antigens.
Parasitol Res. 2001 May;87(5):396‐404.
Cellular immune responses in mice immunized with Anisakis
simplex larval antigens.
The immunization induced a high proportion of CD4+ αβ+ T cells.
The number of CD45+ αβ‐ cells in pre‐immunized and infected mice was lower.
The number of CD4+ T cells increased in infected mice.
In pre‐immunized and infected mice, a decrease of CD8a+ T cells was noted.

8. ESTUDIO DEL EFECTO DE LOS ANTÍGENOS LARVARIOS DE Anisakis simplex SOBRE CÉLULAS
DENDRÍTICAS DERIVADAS DE MÉDULA ÓSEA DE RATÓN
24 h
CITOMETRÍA DE FLUJO
GM‐CSF
RPMI 1640
BMDDCs
In vitro
48 h
IL‐10
IL‐10
IL‐10
IL‐10IL‐10
IL‐10
IL‐10
ELISA: IL‐10
en sobrenadantes
Día 6
de maduración
+
Productos de excreción‐secreción (ES)
Antígeno total (CE)
y/o
Día 6 y 9
de maduración
LPS E. coli 026B6 o CpG (ODN1826),
IL‐10
IL‐12
TNF‐α
Zamora V, Rodero M, Andreu‐Ballester JC, Mendez S, Cuéllar C. Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic
cells. Parasite Immunol. 2019 Apr;41(4):e12616.

9. 43
49
38
65
72 67 70
61 56
CD86
C57BL/6J (% pos)
α
*
β β
*
α
Zamora V, Rodero M, Andreu‐Ballester JC, Mendez S, Cuéllar C. Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic
cells. Parasite Immunol. 2019 Apr;41(4):e12616.
Expression of surface molecules on mouse CD11c+ BMDDCs.
BALB/c (% pos)
* * *
α
The larval antigens of A simplex gave rise to DCs with a “semi‐mature” phenotype, characteristic of the
response to helminth type Th2, with low expression of markers of surface activation.

10. C57BL/6J
18 8 8 11 12 7 7 7
32
26 27 30 30
38
31 33
0
10
20
30
40
50
60
70
ES CE LPS LPS+ES LPS+CE CpG CpG+ESCpG+CE
CD11c+ IL‐10+ (% pos)
12 h 24 h
* *
Zamora V, Rodero M, Andreu‐Ballester JC, Mendez S, Cuéllar C. Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic
cells. Parasite Immunol. 2019 Apr;41(4):e12616.
Expression of intracellular IL‐10 in mouse CD11c+ IL‐10+ BMDDCs.
BALB/c
7 7 4 4 3 5 5 4
12 13
15 14 14
19
15
21
0
5
10
15
20
25
30
35
ES CE LPS LPS+ES LPS+CE CpG CpG+ESCpG+CE
CD11c+ IL‐10+ (% pos)
12 h 24 h
* *
ES and CE induce immature DCs that produce IL‐10.
The intracellular IL‐10 expression drops after 24 hours.
The intracellular IL‐10 expression is associated with lower percentages of CD11c+ IL‐10+ cells.
C57BL/6J showed high levels of CD11c+ IL‐10+ cells.

11. C57BL/6J
10 11
32 35 36
83 83 79
43
49 53 58
79 77 78
84
0
20
40
60
80
100
ES CE LPS LPS+ES LPS+CE CpG CpG+ESCpG+CE
CD11c+ IL‐12+ (% pos)
12 h 24 h
* *
Zamora V, Rodero M, Andreu‐Ballester JC, Mendez S, Cuéllar C. Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic
cells. Parasite Immunol. 2019 Apr;41(4):e12616.
BALB/c
13 14
69 68 68
97 96 97
54 53 55 60 56
63 65 70
0
20
40
60
80
100
ES CE LPS LPS+ES LPS+CE CpG CpG+ESCpG+CE
CD11c+ IL‐12+ (% pos)
12 h 24 h
*
*
*
ES and CE promote inflammatory DCs that synthesize the IL‐12.
The intracellular IL‐12 expression drops after 24 hours.
The intracellular IL‐12 expression is associated with higher percentages of CD11c+ IL‐12+ cells.
BALB/c shows higher intracellular IL‐12 expression.
Expression of intracellular IL‐12 in mouse CD11c+ IL‐12+ BMDDCs.

12. ESTUDIO DEL EFECTO DE LOS ANTÍGENOS LARVARIOS DE Anisakis simplex SOBRE CÉLULAS
DENDRÍTICAS DERIVADAS DE MÉDULA ÓSEA DE RATÓN
Zamora V, Rodero M, Andreu‐Ballester JC, Mendez S, Cuéllar C. Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic
cells. Parasite Immunol. 2019 Apr;41(4):e12616.
ES/CE
BMDDC
madura
CD CD11c+ ¿tolerogénica? CD CD11c+ ¿proinflamatoria?
• Expresión moderada de
CD80/CD86 y MHC II.
• Producción de citocinas
inmunosupresoras como IL‐10 e
inhibir la de citocinas
proinflamatorias como IL‐12 y
TNF‐α.
• Expansión de linfocitos Th2 y T
reguladores.
• Expresión moderada de
CD80/CD86 y MHC II.
• Producción de citocinas
proinflamtatorias como IL‐12 o
TNF‐α .
• Expansión de linfocitos Th1.
IL‐10
IL‐12
TNF‐α

13. EFECTO DE LOS ANTÍGENOS LARVARIOS DE Anisakis simplex SOBRE LA SEÑALIZACIÓN CELULAR
DE CÉLULAS DENDRÍTICAS DERIVADAS DE MÉDULA ÓSEA DE RATONES BALB/c y C57BL/6
Agonista de TLR x + ES/CE
Pam3CSK4
TLR 1‐2 + ES/CE
HKLM
TLR 2 + ES/CE
Poly (I:C) LMW
TLR 3 + ES/CE
LPS E.coli
TLR 4 + ES/CE
CpG (ODN1826)
TLR 9 + ES/CE
ssARN40
TLR 7 + ES/CE
Incrementó
IL‐12, TNF‐α
MHC I
Disminuyó
IL‐10, TNF‐α
Disminuyó
CD11c+ MHC II+
CD11c+ MHC I+ MHC II+
Expresión MHC II
Incrementó
IL‐10+ (12 h)
Incrementó
La expresión de TNF‐α
Incrementó
MHC I, y disminuyó IL‐12,
TNF‐α y NF‐κβ
Incrementó
MHC I, CD80, CD86 , TNF‐α
Incrementó
TNF‐α y NF‐κβ, y disminuyó
IL‐12, MHC I, MHC II
Disminuyó
NF‐κβ
Disminuyó Myd88
BALB/c C57BL/6
BALB/c
respuesta aguda e inflamatoria
C57BL/6J
respuesta más discreta y resistente

14. Expansion of T regulatory lymphocytes by murine bone marrow
dendritic cells previously stimulated with Anisakis simplex larval
antigens

15. CE ↑ CD4+ CD25‐ Foxp3+
CE ↑ CD8+ CD25‐ Foxp3+
CE ↑ CD4+ CD25+ Foxp3+
CE ↑ CD8+ CD25+ Foxp3+
ES ↑ CD4+ CD25+ Foxp3+
ES ↑ CD8+ CD25‐ Foxp3+
Percentages of T cell subsets after 48 hours of co‐culture of mice splenocytes and BMDDCs previously
stimulated with A. simplex larval antigens (ES/CE). Panel A C57BL/6J. Panel B: BALB/c. Percentages of
CD4+ CD25+ or CD4+ CD25‐ and CD8+ CD25+ or CD8+ CD25‐ which express Foxp3. Data are expressed as
mean ± standard deviation of data from three independent experiments. Asterisk indicates statistically
significant differences respect to the control, *P < 0.05.
% Foxp3 C57BL/6J
0 20 40 60
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25-
Control
ES
CE
A
*
*
*
*
*
*
% Foxp3 BALB/c
0 10 20 30
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25-
B
*
*
*
*

16. % IL-10+ C57BL/6J
0 5 10 15 20 25
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25-
Control
ES
CE
A
*
% IFN- BALB/c
0 10 20 30 40
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25-
D
*
*
*
Percentages of T cell subsets after 48 hours of co‐culture of mice splenocytes and BMDDCs previously
stimulated with A. simplex larval antigens (ES/CE). Panels A and B: C57BL/6J. Panels C and D: BALB/c.
Percentages of CD4+ CD25+ or CD4+ CD25‐ and CD8+ CD25+ or CD8+ CD25‐ which express IL‐10 or IFN‐γ.
Data are expressed as mean ± standard deviation of data from three independent experiments. Asterisk
indicates statistically significant differences respect to the control, *P < 0.05.
% IL-10+ BALB/c
0 5 10 15
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25-
*
C
*
*
*

17. Ratio IFN- +/IL-10+ C57BL/6J
0 2 4 6
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25-
Control
ES
CE
A
*
*
*
Ratio of IFN‐γ/IL‐10 expression in CD4+ CD25+, CD4+ CD25‐, CD8+ CD25+ and CD8+ CD25‐ T cell subsets
after 48 hours of co‐culture of mice splenocytes and BMDDCs previously stimulated with A. simplex larval
antigens (ES/CE). Panel A: C57BL/6J. Panel B: BALB/c. Data are expressed as mean ± standard deviation of
data from three independent experiments. Asterisk indicates statistically significant differences respect to
the control, *P < 0.05.
CE  CD8+ CD25+
CE  CD4+ CD25‐
CE  CD4+ CD25+
ES  CD4+ CD25+
Ratio IFN- +/IL-10+ BALB/c
0 10 20 30 40
CD4+CD25+
CD4+CD25-
CD8+CD25+
CD8+CD25- *
B
*
*
*
*
*
*

18. CD4+/CD8+CD25+FOXP3+
IL‐10
IL‐10 IL‐12IL‐12
CD4+/CD8+CD25+IFN‐+
IFN‐
CD4+/CD8+CD25‐FOXP3+ CD4+/CD8+ CD25‐IFN‐ +
IFN‐
- -
--
ESTUDIO DE LA CAPACIDAD DE LAS CÉLULAS DENDRÍTICAS ESTIMULADAS CON LOS PRODUCTOS
LARVARIOS DE Anisakis simplex PARA GENERAR LINFOCITOS T REGULADORES EN RATONES
BALB/c y C57BL/6

19. Anti‐Anisakis simplex IgE: link between allergy and parasitology
Daschner A, Cuéllar C, Rodero M. The Anisakis allergy debate: does an evolutionary approach help? Trends Parasitol. 2012 Jan;28(1):9‐15.

20. A previous contact with Anisakis simplex antigens is
associated with higher IL‐10 and TGF‐β levels in GAA patients.
Daschner A, Rodero M, DE Frutos C, Valls A, Vega F, Blanco C, Cuéllar C. Different serum cytokine levels in chronic vs. acute Anisakis simplex
sensitization‐associated urticaria. Parasite Immunol. 2011 Jun;33(6):357‐62; Daschner A, Fernández‐Fígares V, Valls A, de Frutos C, Rodero M,
Ubeira FM, Cuéllar C. Different fish‐eating habits and cytokine production in chronic urticaria with and without sensitization against the fish‐
parasite Anisakis simplex. Allergol Int. 2013 Jun;62(2):191‐201.
TH1/TH2/TH17 PATTERNS IN ANISAKIOSIS PATIENTS
IL‐2
IL‐4
IL‐6
IL‐10
TNF‐α
IFN‐γ
IL‐17A
TGF‐β

21. Daschner A, Fernández‐Fígares V, Rodero M, Valls A, De Frutos C, Ubeira FM, Cuéllar C. Specific IgG4: possible role in the pathogenesis and a new
marker in the diagnosis of Anisakis‐associated allergic disease. Scand J Immunol. 2014 Feb;79(2):120‐6.
Frequency of occurrence of positive (POS) or
negative (NEG) Ani s 1 and Ani s 7 in gastro‐
allergic anisakiasis (GAA) and A. simplex‐
sensitization‐associated chronic urticaria (CU+).
Specific IgG4 has been associated with protection even when
specific IgE is present.
IgG4/IgE ratios for Ani s 1, Ani s 7 and Anisakis
total larval antigen. Gastro‐allergic anisakiasis
(GAA) and Anisakis‐sensitization‐associated
chronic urticaria (CU+).
The IgG4/IgE‐Ani s 7 ratio was higher in GAA than in CU+.
Ani s 7‐IgG4 is a protection factor and a marker of GAA.
Gastro‐allergic anisakiosis (GAA) patients showed higher IgE and IgG4 levels
against crude extract and both recombinant allergens (Ani s 1 and Ani s 7)
than Anisakis‐sensitization‐associated chronic urticaria (CU+) patients.

22. Supali T, Verweij JJ, Wiria AE, Djuardi Y, Hamid F, Kaisar MM, Wammes LJ, van Lieshout L, Luty AJ, Sartono E, Yazdanbakhsh M. Polyparasitism and
its impact on the immune system. Int J Parasitol. 2010 Aug 15;40(10):1171‐6.

23. Existe un sinergismo entre ambos parásitos con potenciación de la urticaria crónica cuando se
presenta una asociación positiva de la infección crónica por Toxoplasma gondii con el
parasitismo previo por Anisakis simplex.
Fernández‐Fígares V, Rodero M, Valls A, De Frutos C, Daschner A, Cuéllar C. Positive associations between infections of Toxoplasma gondii and
seropositivity with Anisakis simplex in human patients suffering from chronic urticaria. J Helminthol. 2015 Nov;89(6):707‐13.

24. • Prevalencia de la infección por Toxoplasma gondii. La
infección por Toxoplasma gondii se determinó por
ELISA. CU+: urticaria crónica (CU) asociada a sensibilización
a Anisakis simplex. CU‐: CU sin sensibilización a A. simplex.
 Prevalencia de atopia en función de la infección
por Toxoplasma gondii en pacientes con urticaria crónica. La
atopia se define como la positividad de al menos
un aeroalergeno (polen, moho, caspa de animales o ácaros del
polvo doméstico), independiente de la sensibilización
a Anisakis simplex. Tg‐: no T. gondii infección. Tg+:
seropositividad indica infección por T. gondii
p=0,014
Tg- Tg+
p= 0,08
Fernández‐Fígares V, Rodero M, Valls A, De Frutos C, Daschner A, Cuéllar C. Positive associations between infections of Toxoplasma gondii and
seropositivity with Anisakis simplex in human patients suffering from chronic urticaria. J Helminthol. 2015 Nov;89(6):707‐13.
Se observó una inesperada asociación positiva entre la infección crónica por Toxoplasma gondii medida mediante
determinación de IgG y la IgE específica anti‐Anisakis simplex como marcador de parasitismo previo por este
nematodo.
La sensibilización frente a aeroalergenos se asoció con la infección crónica por T. gondii.

25. Anti‐Anisakis sp. antibodies in serum of healthy subjects.
Relationship with αβ and γδ T cells
Zamora V, García‐Ballesteros C, Benet‐Campos C, Ballester F, Cuéllar C, Andreu‐Ballester JC. Anti‐Anisakis sp. antibodies in serum of healthy
subjects. Relationship with αβ and γδ T cells. Acta Parasitol. 2017 Mar 1;62(1):97‐103.
Correlations among NKT cells and anti‐Anisakis antibody levels
‐ All the specific isotypes studied were negatively correlated with NKT cell rates with the exception of IgG.
‐ CD19 expression levels correlated positively with serum specific IgM.
Previous contact
NKT decrease

26. Significant frequency of T cell subsets according to positive (P) and negative
(N) total immunoglobulins (Ig’s), IgA and IgM anti‐Anisakis antibodies.
The anti‐Anisakis positivity
was related with a decrease
of CD56+αβ+ and all γδ+ T
cell subsets especially in the
case of anti‐Anisakis IgA.
Zamora V, García‐Ballesteros C, Benet‐Campos C, Ballester F, Cuéllar C, Andreu‐Ballester JC. Anti‐Anisakis sp. antibodies in serum of healthy
subjects. Relationship with αβ and γδ T cells. Acta Parasitol. 2017 Mar 1;62(1):97‐103.

27. Anti‐Anisakis sp. antibodies in serum of patients with sepsis and their
relationship with γδ T cells and disease severity
Andreu‐Ballester JC, Tormo‐Calandín C, Garcia‐Ballesteros C, Pérez‐Griera J, Amigó V, Almela‐Quilis A, Ruiz del Castillo J, Peñarroja‐Otero C,
Ballester F. Association of γδ T cells with disease severity and mortality in septic patients. Clin Vaccine Immunol. 2013 May;20(5):738‐46; Andreu‐
Ballester JC, Zamora V, Garcia‐Ballesteros C, Benet‐Campos C, Lopez‐Chuliá F, Tormo‐Calandín C, Cuéllar C. Anti‐Anisakis sp. antibodies in serum of
patients with sepsis and their relationship with γδ T cells and disease severity. Int J Parasitol. 2018 May;48(6):483‐491.
Deficiency of αβ T cell (A) and γδ T
cell (B) subsets according to the
severity of sepsis in patients
(percentages).
γδ T cells were related to the
severity and poor prognosis of
septic patients

28. Andreu‐Ballester JC, Zamora V, Garcia‐Ballesteros C, Benet‐Campos C, Lopez‐Chuliá F, Tormo‐Calandín C, Cuéllar C. Anti‐Anisakis sp. antibodies in
serum of patients with sepsis and their relationship with γδ T cells and disease severity. Int J Parasitol. 2018 May;48(6):483‐491
(A) Total IgS: control‐sepsis
and severe sepsis, P = 0.001;
sepsis and septic shock, P <
0.0001.
IgM: control‐sepsis and
severe sepsis, P = 0.016;
control‐sepsis and septic
shock, P = 0.002.
IgA: control‐sepsis and
severe sepsis, P = 0.004;
control‐sepsis and septic
shock, P < 0.0001.
(B) IgE: septic shock and
others, P = 0.003.
(C) IgG: septic shock and
others, P = 0.038.
(D) positive (%) IgG anti‐
Anisakis
Levels of patients’ anti‐Anisakis sp. antibodies according to
severity of sepsis

29. Andreu‐Ballester JC, Zamora V, Garcia‐Ballesteros C, Benet‐Campos C, Lopez‐Chuliá F, Tormo‐Calandín C, Cuéllar C. Anti‐Anisakis sp. antibodies in
serum of patients with sepsis and their relationship with γδ T cells and disease severity. Int J Parasitol. 2018 May;48(6):483‐491
The decreases in anti‐
Anisakis IgA levels were
associated with a delayed
admission to hospital, and
severity of sepsis, as well as
an increase in APACHE and
SOFA scores.
There is a direct relationship
between levels of anti‐Anisakis
IgA and γδ T cells
The lack of protection in the
mucosa (IgA and γδ T cells)
was associated with the
disease severity.

30. Juan González-Fernández
Marta Rodero
Virginia Fernández-Fígares
Vega Zamora
Alvaro Daschner
Consolación de Frutos
Ana Valls
Teresa Gárate
Esperanza Rodríguez
Maria Jesús Perteguer
Florencio M. Ubeira
Ana Anadón
Fernanda Romarís
Carmen del Águila
Soledad Fenoy
Tomás Chivato
Luis Rivas
Juan Román Luque Ortega
Andreas Lopata
Natalie E. Nieuwenhuizen
Juan Carlos Andreu-Ballester
Carlos García-Ballesteros
Carmen Benet-Campos
Ferrán Ballester
Rafael Gil-Borrás
Ignacio Catalán-Serra
Christiane Kruse Fæste