Inflammatory testicular lesions 2 - Friday/3 Urogenital... · Male Infertilitiy Microbiology and Inflammation in Urogenital Infections (MIBIE) Inflammatory testicular lesions Martin

Male Infertilitiy Microbiology and Inflammation in Urogenital Infections (MIBIE)

Inflammatory testicular lesions

Martin Bergmann Thomas Haggeney, Britta Klein, and Hans-Christian Schuppe

Summer School 2014, 31st of July -2nd of August

Immunprivilege of the testis


Extended survival of cells expressing antigens that under normal conditions should provide immune response Established in puberty, when Blood-Testis-Barrier (BTB) is formed and new antigens appear with the initiation of spermatogenesis.

adluminal compartment Spermatocytes, spermatids

BTB

Mechanisms: BTB as anatomical, physiological and immunological barrier leads to the reduction of interaction between germ cells and immune cells

basal compartment Spermatogonia, preleptotene permatocytes

TGF-ß

Local secretion of immune suppressive, i.e.: anti-inflammatory cytokines such as TGF-ß and IL-10

IL-10

adapted from Perez et al. (2013) Dual role of immune cells in the testis. Spermatogenesis 3:1, e23870


Testicular inflammation: classification

Epididymo-orchitis ("bacterial" or "unspecific" orchitis)

ascending, canalicular infections - STD = C. trachomatis, N. gonorrhoeae [< 35 J.]

- UTI = E. coli (and other Enterobacteriaceae) [> 35 J.]

adapted from Schuppe and Bergmann, 2012


Epididymo-orchitis (bacterial)

Interstitial edema, lenlarged blood vessels and massive infiltration of seminiferous tubules and interstium with lymphocytes and granulocytes

adapted from Köhn et al. (2005)






Orchitis associated with systemic infections ("viral orchitis")

systemic viral infections - Mumps, Coxsackie virus, HIV, CMV, Influenza, Parainfluenza, Mononucleosis, Varicella, Echo- &

Enterovirus, Dengue fever …



Orchitis associated with systemic infections ("viral“)

Sequelae of mumps orchitis with persistence of focal inflammation consiting of dense peritubular lymphocytic infiltrates. Meshwork pattern of affected lamina propria and disruption of the seminiferous epithelium

adapted from Schuppe and Bergmann (2013)









Predominantly granulomatous orchitis ("specific" orchitis) - Tbc, Syphilis, Lepra, Brucellosis

Chronic /idiopathic granulomatous orchitis of unknown origin [> 50 J.



Subacute granulomatous orchitis

Dense inflammatory infiltrate, mainly consisting of lymphocytes, with focal necrosis surrounded by epitheloid cells (macrophages) and residual tubular structures










Predominantly granulomatous orchitis ("specific" orchitis) - Tbc, Syphilis, Lepra, Brucellosis

Chronic /idiopathic granulomatous orchitis of unknown origin [> 50 J.



Chronic idiopathic granulomatous orchitis

Dense peritubular lymphocytic infiltrate

withlymphocytes, macrophages and

plasma cells.

Note complete atrophy of seminiferous

epithelium in adjacent seminiferous

tubules („tubular shadows“)



Asymptomatic focal inflammatory

lesions in infertile men

T- Lymphocytes

Macrophages

from: Bergmann M, Kliesch S (2010)


Pattern analysis of lymphocytic infiltrates

dense sparce scattered intratubular macrophages


Prevalence of lymphocytic infiltrates

n=91 unilateral bilateral

Negative 27

Infiltrate + scattered 23 12

sparse 11 3

dense 9 6

(in 48 % of the affected testes lesions in >1 site)


Pattern analysis of lymphocytic infiltrates Relationship to spermatogenic impairment

Score count analysis

left 2

scattered

from: Bergmann M, Kliesch S (2010)


left 2


Score count analysis


% of biopsies

p < 0.001



„Players involved“

surface marker (Cluster of Differentiation) Lymphocytes T- Lymphocytes CD3 (pan) B-Lymphocytes CD 20cy Antigen presenting cells Macrophages CD68 Dendritic cells CD11c (also expressed by macrophages) S-100 Mast cells tryptase Natural killer cells CD56 (not found) Neutrophil granulocytes detected by typical nuclear structure



T

T

CD3 T-cells

T

T

CD20cy B-cells

T

T

CD68 macrophages

T

T

CD11c dendritic cells

T

T

Mast cells (tryptase)

T

T

HE


Cytokines/Chemokines

Testicular somatic cells Sertoli cells: IL-1ß, IL-6, TNF-α, TGF-ß Leydig cells: Il-6, TGF-ß, IFN-γ Peritubular cells: TNF-α, IL-6, IFN-γ Macrophages: TNF-α, IL-10, Il-12, TGF-ß Mast cells: TNF-α

Regulatory Inflammatory

APC´s: Antigen-presenting cells (macrophages /dendritic cells)

Th17

Th2 effector

Th1 effector

Apc`s Il-12, IL-18

Treg

IFN-ϒ

Il-23 Il-4, IL-5, IL-13

IL-10, TGF-ß IL-10, TGF-ß

IL-6 TGF-ß1

IL-17A, IL-21, IL-22

Effector T cells Message: Cytokine environment/ network


Duan et al. (2011) Immunodeviation towards a Th 17 immune response associated with testicular damage in azoospermic men. Int J Androl 34:e536-e545 Increase of CD3 T-lymphocytes producing IL-17A CD3 IL-17A

CD68 IL-23

Increase of CD68 and CD11c APCs producing IL-23




Th17

Th2

Th1 Apc`s

Il-12, IL-18

Treg

IFN-ϒ

IL-23 Il-4, IL-5, IL-13

IL-10, TGF-ß IL-10,TGF-ß1

IL-6 TGF-ß1

IL-17A, IL21, IL-22

Duan et al. (2011) Immunodeviation towards a Th 17 immune response associated with testicular damage in azoospermic men. Int J Androl 34:e536-e545 „From these data, a picture emerges that Th17 cells orchestrated by IL-23 producing APCs are critically involved in chronic inflammation in ATCI.“ ACTI = Azoospermic Testis with Chronic Inflammation

Inflammatory Regulatory


IL-α IL-1 IL-2 IL-5 IL-6 IL10 IL-12A IL-12B IL-13 IL-17A IL-20 (part of IL-23) Il-23a INF-ϒ TNF-α TGF-ß CxCl- 10, 13, CCL5

Cytokines/Chemokines analysed by qualitative RT-PCR

M

IFNγ

C M

IL1β

C

IL1

2 p

35

C

IL6

C

TN

Fα

β

-actin

C M


TNF-α

3 2 M neg.

1 3

2

2

3



Cytokine milieu is dominated by anti-inflammatory IL-10 predominantly expressed by testicular macrophages. Normalized to IL-12A, which reflects immune cell expression



Cytokines/Chemokines analysed by quantitative RT-PCR

Increase of pro-inflammatory IL-1ß, IL6, IFN-ϒ and TNFα produced by testicular somatic cells associated with spermatogenic impairment.

No real difference in samples containing focal lymphocytic infiltrates.


• Infiltration of immune cells, i.e. T-lymphocytes, seen in testes of

infertile men reflects a profound disturbance of the local

immunoregulation (immune privilege).

• Focal or multi-focal, mainly peritubular localisation of effector T

cells is associated with characteristic alterations of the affected

seminiferous tubules as well as overall deterioration of

spermatogenesis.

Hypothesis:

• Spermatogenic impairment of unknown etiology promotes a pro-

flammatory environment produced by testicular somatic cells,

with subsequent formation of (focal) inflammatory lesions.

Conclusions I


Inflammation associated with testicular cancer

Dieckmann and Huland, 2001

Genesis of germ cell tumors

TIN/ TIN

TIN

TIN

TIN

Seminoma

adapted from: Donner et al., 2004


Hvarness et al. (2013) Phenotypic characterisation of immune cell infiltrates in testicular germ cell neoplasia. J Reprod Immunol 100:135-145

Message: „This study showed a pheno-typically comparative compo-sition of infiltrating immune cells independentely of the presence of neoplasia, suggesting the absence of active surveillance in testicular cell cancer.“


nsp

Hyp + Ly

TIN + Ly

Seminom + Ly


TIN „Players involved“

CD68 macrophages CD11c dendritic cells Mast cells (tryptase)

HE CD3 T-cells CD20cy B-cells Involvement of

B-lymphocytes

and

dentritic cells


Seminoma „Players involved“

Mast cells (tryptase)

CD3 T-cells HE

CD11c dendritic cells CD68 macrophages

CD20cy B-cells Follicular-like

structures

Involvement of

B-lymphocytes

and

dendritic cells


Increase of pro-inflammatory cytokines, especially IL-6 and TNF-α Is in line with the involvement/ detection of CD20cy B-cells

**

***

*

*

**


Increase of TGF-ß Most probably due to the large numbers of Apc`s

*

**


Increase of IL-2 and INF-ϒ Hypothesis: Possible answer to tumor antigens.

*

***


Decrease of Th-2 cytokines (IL5, 13, 23a)

***

*** ***

n.d. n.d.

Th1

Th2 IL-4

IL-5

IL-1

IL-23a

IL-2

IL-12

IFN-

The balance of Th1 and Th2 switches to Th1 i.e.: in the case of neoplasia, there is a Th1 driven immune response


Increase of CxCl´s is in line with the recruitment of CD20cy B-cells and follicular-like structures

**

**

**

***

*


• In testicular neoplasia, infiltration of T-lymphocytes is accompanied by large

numbers of antigen-presenting cells (macrophages, dendritic cells; APC) as

well as B cells.

• Pattern and high levels of pro-inflammatory cytokines/chemokines produced

by these cells are well in line with an immune response driven by Th1 cells

and a prominent role of B cells (formation of follicle-like structures).

Hypothesis:

• Along with testicular neoplastic processes, a tumor-specific immune response

is initiated for actively controlling further tumor progression.

Conclusions II

Documents

Inflammatory testicular lesions 2 - Friday/3 Urogenital... · Male Infertilitiy Microbiology and Inflammation in Urogenital Infections (MIBIE) Inflammatory testicular lesions Martin