Center fo Research and Advanced Studies, National Polytechnic Institute

Department of Physiology, Biophysics and Neurosciences, Mexico, City

Pulmonary tuberculosis and male reproductive system: an immunoendocrine approach.

Marta C. Romano Pardo

Pulmonary tuberculosis

Scanning Electron Micrograph of Mycobacterium tuberculosis

Gram-Positive Bacillus Strict aerobic Immobile 4µ of length Bacillus Acid Alcohol

Resistant (BAAR)

Zumla et al., 1999; Bottasso et al., 2007; Del Rey et al., 2007)

(WHO, 2016)

Global incidence of tuberculosis in 2014. Map obtained from Global tuberculosis (Report 2015)

Pulmonary tuberculosis

Tuberculosis is the leading cause of death in the world by an infectious agent.

In 2015, 10.4 million people became ill with tuberculosis and 1.8 million died from tuberculosis

More than 95% of tuberculosis deaths occur in low and middle income countries, with six countries accounting for 60% of total mortality; India, followed by Indonesia, China, Nigeria, Pakistan and South Africa

Tuberculosis is one of the leading causes of death in HIV-positive people: by 2015, 35% of HIV-related deaths were due to tuberculosis

Statistics indicate that the incidence of tuberculosis has declined by 1.5% per year since 2000

It is estimated that one third of the world population has latent tuberculosis, which means that there are people infected by the bacillus but who have not yet become sick and can not transmit the infection

(WHO, 2017)

Pulmonary tuberculosis

Immune response

Nutritional status

Pregnancy

Gender

(SSA, 2016)

Productive cough (sometimes with blood in the sputum)

(WHO, 2016;SSA, 2016)

Chest pains

Weakness

Weight loss

Fever

Night sweats

SYMPTOMS

Endocrine response and cytokines in pulmonary tuberculosis

(Dlugovitzky et al., 1997; Condos et al., 1998; Del Rey et al., 2007)

Simulate the development of delayed-type

hypersensitivity reactions and the activation of

macrophages

(Da silva, 1999; Bottasso et al., 2007)

The endocrine response is parallel to the immune

response on the tuberculosis bacillus

(Krameer and Wray., 2002; Del Rey et al., 2007)

(Bottasso et al., 2007; Del Rey et al., 2007)

Hypothalamic-pituitary-adrenal-gonadal and thyroid

axes

(Bottasso et al., 2007; Brown et al., 2010)

Facilitates Th2 activity Inhibits Th1 activity

IFN-γ. IL-10 and lL-6

Testosterone and Dehidroepiandrosterone

(DHEA)

(Del Rey et al., 2007; Bozza et al., 2007; Fiorenza et al., 2008)

Participation of the hypothalamic-pituitary-adrenal axis in the immunopathology of tuberculosis

GLUCOCORTICOIDS

CRH

ACTH

CEREBRAL CORTEX

IL-1 IL-6 TNF

DHEA (Rook G et al., 1997; Rook G et al., 2000)

Tuberculosis and reproductive aspects

(Kumar, 2008)

Genital tuberculosis Pulmonary tuberculosis

Tuberculous epididymo-orchitis

Decreased testosterone and DHEA

concentrations

(Del Rey et al, 2007)

Objective

• The aim of this study was to investigate whether pulmonary TB produces immunoendocrine alterations in the male mice reproductive system.

Infected with M. tuberculosis (H37Rv)

Without M. tuberculosis

infection

Days of sacrifice 1,3,7,14,21,28,60,90 y 120 PI

Male mice BALB/c

Blood (serum)

Testosterone

RIA

Testes Epididym

CFU

Histology

Lungs

Prostate gland

Seminal vesicle

Testes

Spermatobioscopy

Viability

Motility

Sperm Capacitation

Sperm Count

IL-1β, IL-6, IL-

10, TNF-α, INF-γ, TGF-β

Inmuno-histochemistry

Real time-PCR

Presence of bacteria in tissues: CFU

Figura 2. Determinación de la carga bacilar en homogenizados

pulmonares de ratones machos infectados con H37Rv. Cada

barra representa la media ± DE de 2 experimentos

independientes (n=6 por grupo).

The detection of bacteria in extrapulmonary tissue was

negative

1 3 7 14 21 28 60 90 120

0

20

40

60

80

Post-infection days

Lu

ng

Bacilli L

oad

s (

CF

U 1

x10

6)

Pulmonary bacilli loads of male mice. Mice were infected by intratracheal route with Mycobacterium tuberculosis H37Rv and were sacrificed at the indicated days after infection. The lungs (two independent experiments n=4 lungs per group) were used for determination of colony forming units. At late disease, male mice showed significant higher bacilli loads.

Organ/ Body weights

CONTROL H37Rv0

10

20

30CONTROL

H37Rv*

A)L

un

g w

eig

ht

(mg

)

CONTROL H37Rv0

10

20

30

40

*

B)

H37Rv

CONTROL

Lu

ng

weig

ht

(mg

)

TESTIS

SEM

INAL V

ESIC

ULE

PROSTA

TE GLA

ND

0

2

4

6control

H37Rv*

**

*

c)

Org

an

weig

ht

(mg

)

TESTI

S

SEM

INAL

VESIC

LE

PROSTA

TE G

LAND

0

2

4

6 CONTROLH37Rv*

* *

*

D)

Org

an

weig

ht

(mg

)

Organ/body weights. A). Lung to

body weight at 90 days post-

infection. B. Lung to body weight

at 120 days post-infection. C, D.

Testis, seminal vesicles and

prostate weights corrected by

body weights at days 90 and 120

post-infection respectively.

Determination of serum testosterone concentrations in mice with non disseminated pulmonary tuberculosis

D1

D3

D7

D14

D21

D28

D60

D90

D12

0

0

50

100

150

200CONTROL

H37Rv

*

Post-infection days

Testo

ste

ron

e c

on

cen

trati

on

s

in

seru

m (

pg

/ml)

Serum testosterone concentrations (pg/ml) of control and H37Rv infected mice. Date are expressed as mean and standard deviation of two independent experiments, 6 animals each day of sacrifice.

Histopathology of seminal vesicles of mice with ND pulmonary tuberculosis

CONTROL H37Rv

D1

20

D

90

D

60

Seminal vesicle epithelium of healthy and H37Rv infected mice. A) and B). Height and thickness of seminal vesicle cells. Each bar represents the mean ± SD of two independent experiments (n=3-6 per group, P<0.05). C). Representative micrographs of seminal vesicle from control non-infected mouse (Left) showing well preserved cilindrical epithelium and infected mouse (right) that show cubical atrophic epithelium at days 60, 90 and 120 post-infection. Hematoxilin/eosin staining, 40X magnification.

1 3 7 14 21 28 60 90 120

0

5

10

15

20

CONTROL

H37Rv

* *

Post-infection Days

A)

Cells h

eig

ht

( m

)

1 3 7 14 21 28 60 90 120

0

5

10

15

20 CONTROL

H37Rv

Post-infection days

B)

Cells t

hic

kn

ess (

m)

Histopathology of prostate gland of mice with ND pulmonary tuberculosis

CONTROL H37Rv

D1

20

D

90

D

60

1 3 7 14 21 28 60 90 120

0

5

10

15

20CONTROL

H37Rv

* **

Post-infection days

A)

Cells h

eig

ht

( m

)

1 3 7 14 21 28 60 90 120

0

5

10

15CONTROL

H37Rv

*

Post-infection days

B)

Cells t

hic

kn

ess (

m)

Prostate epithelium of

healthy and H37Rv infected

mice. A) and B). Height and

thickness of prostate cells.

Each bar represents the mean

± SD of two independent

experiments (n=3-6 per

group, P<0.05). C).

Representative micrographs

of a prostate from healthy

(Left) and infected mice

(right) mice stained with H &

E at days 60, 90 and 120 post-

infection. 40X.

Spermatozoid Count

Figura 18. Determinación de la viabilidad espermática en machos infectados

con H37Rv y ratones sanos. Cada barra representa la media ± DE de 2

experimentos independientes (n=6 por grupo).

1 3 7 14 21 28 60 90 120

0

20000

40000

60000

80000

CONTROLH37Rv

* *

*

Post-infection days

Nu

mb

er

of

sp

erm

cells

Spermatozoid number of mice infected with H37Rv. Number of spermatozoids in infected and healthy animals. Each bar represents the mean ± SD of two independent experiments (n=3-6 per group).

Testicular Interleukine-6 (IL-6) : Immunohistochemistry and Real Time PCR

Figura 18. Determinación de la viabilidad espermática en machos infectados

con H37Rv y ratones sanos. Cada barra representa la media ± DE de 2

experimentos independientes (n=6 por grupo). 21 28 60 120

0

100000

200000

300000

400000

500000CONTROL

H37Rv

*

*

Post-infection days

Co

pie

s o

f IL

-6/

10

5 c

op

ies o

f R

LP

OControl H37Rv

D6

0

D9

0

D1

20

A) B)

Representative immunohistochemistry for IL-6 protein and gene expression in testicular tissue. A) Sections from H37Rv infected and control mice. The tissues were incubated with a specific antibody to detect the indicated cytokine. The signal for IL-6 was found in Sertoli cells (20X). B) Kinetics of IL-6 gene expression by PCR-Real Time in the testis of control and infected mice.

Testicular Transforming Growth Factor Beta(TGF-β): Immunohistochemistry and Real Time PCR

Control H37Rv

D6

0

D9

0

D1

20

A)

21 28 60 120

0

1000

2000

3000

4000

5000CONTROL

H37Rv

* **

Post-infection days

Co

pie

s o

f T

GF

- /

10

5co

pie

s o

f R

LP

O

B)

Representative immunohistochemistry for TGF-β protein and gene expression in testicular tissue. A)

Sections from H37Rv infected and control mice. The tissues were incubated with a specific antibody to

detect the indicated cytokine. The signal for TGF-β was found in Leydig cells (20X). B) Kinetics of TGF-β

gene expression by PCR-Real Time in the testis of control and infected mice.

Testicular Interleukine-10 (IL-10): Immunohistochemistry and Real Time PCR

Control H37Rv

D6

0

D9

0

D1

20

A) B)

21 28 60 120

0

10000

20000

30000

40000CONTROL

H37Rv

* * * **

*

Post-infection days

Co

pie

s o

f IL

-10/1

05

co

pie

s o

f R

LP

O

Representative immunohistochemistry for IL-10 protein and gene expression in testicular tissue. A) Sections from H37Rv infected and control mice. The tissues were incubated with a specific antibody to detect the indicated cytokine. The signal for IL-10 was found in spermatogonia and spermatozoa (20X). B) Kinetics of IL-10 gene expression by PCR-Real Time in the testis of control and infected mice.

Conclusions

• Our results show that non disseminated pulmonary TB affects the male reproductive organs and the sperm production.

• The expression of testicular pro-inflammatory cytokines are also altered.

• The immunological alterations that occur during the course of pulmonary TB may affect the reproductive system by causing an imbalance in androgen production that may affect the function and morphology of their target organs.

M.Sce BRENDA RAMOS Dr. ROGELIO HERNANDEZ PANDO BIOL RICARDO VALDEZ

Gracias…

Body weight of mice with pulmonary tuberculosis

1 3 7 14 21 28 60 90 120

0

10

20

30

40

CONTROL

H37Rv

*

**

Post-infection days

Bo

dy w

eig

ht

(g)

FIGURE 2. Body weight of mice with pulmonary tuberculosis (H37Rv) and control healthy animals. Each

point represents the mean ± SD of two independent experiments (n=6 animals per group). During late

disease significant differences in body weight between H37Rv infected mice and control animals were

found.

Mice lung and sexual organs weights during the course of H37Rv infection

3 7 14 21 28 60 90 120

0

200

400

600CONTROL

H37Rv**

*

**

Post-infection days

A)

Lu

ng

weig

ht

(mg

)

1 3 7 14 21 28 60 90 120

0

100

200

300CONTROL

H37Rv

B)

Post-infection days

Testi

s w

eig

ht

(mg

)

1 3 7 14 21 28 60 90 120

0

50

100

150CONTROL

H37Rv

* **

*

C)

Post-infection days

Sem

inal vesic

le w

eig

ht

(mg

)

1 3 7 14 21 28 60 90 120

0

20

40

60

80

100CONTROL

H37Rv

**

D)

Post-infection days

Pro

sta

te g

lan

d w

eig

ht

(mg

)

Figure 4. Mice lung and sexual

organs weights during the course

of H37Rv infection. A. Lung

weights: A significant increase

was observed in mice lung weight

respect to control animals from

14 days post-infection onwards.

B) No differences between

infected and non-infected mice

testis were found. C). A significant

weight decrease was observed in

the seminal vesicles of infected

related to control mice since 28

days post-infection. D). A

significant decrease was observed

in the prostate gland weights of

infected mice related to control

animals at 90 and 120 days post-

infection.

Spermatobioscopy

1 3 7 14 21 28 60 90 120

0

20000

40000

60000

80000

CONTROLH37Rv

* *

*

A)

Post-infection days

Nu

mb

er

of

sp

erm

cells

1 3 7 14 21 28 60 90 120

0

20

40

60

80

100CONTROLH37Rv

Post-infection days

B)

Perc

en

tag

e c

ell v

iab

ilit

y (

%)

1 3 7 14 21 28 60 90 120

0

20

40

60

80

100CONTROL

H37Rv

Post-infection days

C)

Perc

en

tag

e o

f cell m

oti

lity

(%

)

1 3 7 14 21 28 60 90 120

0

20

40

60

80

100 CONTROLH37Rv

D)

Post-infection days

Perc

en

tag

e o

f sp

erm

cap

acit

ati

on

(%

)

Figure 8. Sperm quality of mice infected with H37Rv. A). Number of spermatozoids in infected and healthy animals. B). Percentage of cell viability in infected mice compared to healthy animals. C). Percentage of cell motility in infected and healthy mice. D). Percentage of sperm capacitation in infected mice compared to healthy animals. Each bar represents the mean ± SD of two independent experiments (n=3-6 per group).