The immune-privilege of the testis – causative mechanism for disturbed spermatogenesis E. coli

epididymitis model, E. coli orchitis model

Sudhanshu Bhushan

1st August 2014

MIBIE Summer School

Aetiology of male infertility

� Idiopathic infertility 31,7%

� Varicocele 6,6%

� Infections (subclinical) 9,0%

� (Endocr), hypogonadism 8,9%

� Cryptorchidism 8,5%

� Disturbance of emission 5,8%

and ejaculation (incl. erectile

dysfunktion, hypospadia etc.)

� Systemic diseases 5,0%

� immunolog. factors 4,2%

� Testis tumor 2,3%

� Obstructions 1,5%

� Others 6,6%

Nieschlag and Behre, 2000

� Infection and immunological infertility: second most important cause with 13-14%

Epidemiology and Pathogens

� A disease of all ages�STD‘s preferentially in young, sexually active patients

n=131

Pilatz et al., unpublished

n=148

Age of patients

STDs

Enterobacteriaceae

No bacterial pathogen

Bacterial Spectrum

�E. coli dominant pathogen

Pilatz et al., unpublished

No bacterial pathogen

E. Coli

Enterococcus spp.

Pseudomonas spp.

S. aureus

Proteus spp.

Morganella spp.

Chlamydia trachomatis

Neisseria gonorrhoeae

Mycoplasma spp.

S. epidermidis

Klebsiella spp.

Bacteria induced epididymo-orchitis model

Bhushan et al., 2009

Caput CaudaC

ontr

ol3d

UP

EC

7d U

PE

C

Testis

1000x1000x

Bacterial induced epididymo-orchitis model: Mouse

Bacterial induced epididymo -orchitis model: Rat

Ctrl UPEC

UPEC CFT073 induces epididymo -orchits

Ctrl testis Infected testis

UPEC CFT073 induces germ cell loss in testis

20x

40x

UPEC infected testis (Electron microscope picture)

UPEC CFT073 induces DNA break in testicular cells

Ctrl

UPEC

Pathways leading to cell death

Fink et al., 2005

Apoptosis signaling pathways

Rudel et al., 2010

UPEC does not induce apoptotic signaling pathways

Extrinsic apoptosis

Intrinsic apoptosis

Pyroptosis

Is UPEC induces necrosis?

Ctrl UPECRaw macrophageC + -

EM picture of UPEC infected testis

HMGB1 translocate to the cytoplasm following infect ion with UPEC

Uropathogenic E. coli (CFT073 and 536) induces cell death in Sertoli cells and peritubular cells

Ctrl NPEC UPEC

Uropathogenic E. coli CFT073 induces cell death in Sertoli cells and peritubular cells

UEPCCtrl NPEC

PTC

SC

Apoptosis signaling pathways

Rudel et al., 2010

Jurkat cell extract- +

PTC0 .5 1 2 3 4 6Time (h)

Caspase 6

Caspase 3

PTC0 .5 1 2 3 4 6Time (h)

SC0 .25 .5 1 2 3

SC0 .25 .5 1 2 3

UPEC CFT073 does not induce intrinsic apoptosis pat hways in Sertoli cells and peritubular cells

UPEC does not induce enzymatic activity of Caspase 3

SC0 1 2 3

PTC RAW ø0 1 2 3 4 6 0 8

Caspase-8

β-Actin

Time (h)

UPEC CFT073 does not induce extrinsic apoptosis pathways in Sc and PTC

M SCCtrl UPEC

PTCCtrl UPECM MRAW ø

Ctrl SNP F/T

DNA laddering was not observed following UPEC infection in Sertoli cells and peritubular cells

Programmed cell necrosis

Kennedy et al., 2008

Sertoli cells Peritubular cells

Calpain activation No yes

ROS production No yes

ATP depletion yes yes

LDH release yes yes

UPEC induces programmed cell necrosis in testicular cells

UPEC induces extracellular release of HMGB1 in in Sertoli cells and peritubular cells

Ctrl UPEC

UPEC induces extracellular release of Histone 3 in Sertoli cells

Ctrl NPEC UPEC

Histone 3

HMGB1

Ctrl NPEC UPEC

Histone 3

HMGB1

PTC

NPEC

SC

Danger signals released from necrotic cell could induce autoimmunity in infected testis

Summary

� Bacteria can reach testis through ascending canicularinfection.

� UPEC infection resulted in severe impairment ofspermatogenesis by germ cell loss, damage oftesticular somatic cell and a decrease in spermnumbers.

� Necrosis appears to be the dominant cell deathpathway in UPEC infected testis. Substantial necroticchanges seen in Sertoli cells will contribute to impairedspermatogenesis by loss of function in supporting thedependent germ cells.

Collaborations

Dept. Urology & Androlog yW. WeidnerF. WagenlehnerH-C. SchuppeA. PilatzK. StegerA. Paradowska-Dogan

Institute of Medical MicrobiologyT. ChakrabortyH. HossainS TchatalbachevE. Domann

Dept. of Anatomy and Cell BiologyA. MeinhardtM. FijakV. MichelF. ÁslaniY. Lu Z. Zhang Institute of Veterinary Anatomy

M. Bergmann