Porphyromonas gingivalis - SciELO

Original Article http://dx.doi.org/10.1590/1678-77572016-0277

7ROOOLNHUHFHSWRUDJRQLVWV Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells Abstract Zhiqiang LIU1,2 Yang HU1 Pei YU1,3 Mei LIN2

,/H[SUHVVLQJUHJXODWRU\%FHOOV% SOD\DNH\UROHLQLPPXQHV\VWHP EDODQFHE\OLPLWLQJH[FHVVLYHLQÀDPPDWRU\UHVSRQVHV(IIHFWVRIWROOOLNHUHFHSWRU signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective: This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and

Grace HUANG1

IL-10 competency in vitro. Material and Methods: Spleen B cells were isolated

Toshihisa KAWAI1

IURP&%/-PLFHZLWKRUZLWKRXWIRUPDOLQ¿[HGP. gingivalis immunization.

Martin TAUBMAN1

B cells were cultured for 48 hours under the following conditions: CD40L,

Zuomin WANG

2

CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of

Xiaozhe HAN

1

¿[HGP. gingivalis. Percentages of CD1dhiCD5+%FHOOVZHUHPHDVXUHGE\ÀRZ cytometry. IL-10 mRNA expression and secreted IL-10 were measured by realtime quantitative PCR and by ELISA respectively. Results: P. gingivalis LPS plus &'/VLJQL¿FDQWO\LQFUHDVHG&'GKL&'%FHOOSHUFHQWDJHVDQGVHFUHWHG,/ 10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels ZHUHVLJQL¿FDQWO\LQFUHDVHGLQ&'//36WUHDWHGJURXSFRPSDUHGZLWK&'/ treatment group in the absence of P. gingivalis&S*SOXV&'/VLJQL¿FDQWO\ decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions: P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses. Keywords: IL-10. Porphyromonas gingivalis LPS.

Submitted: June 3, 2016 0RGL¿FDWLRQ$XJXVW Accepted: September 20, 2016

Corresponding address: Xiaozhe Han 245 First Street, Cambridge, MA 02142 - USA Phone: (617) 892-8447 - Fax: (617) 892-8612 e-mail: [email protected]

1 The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, Massachusetts, United States. 2 Capital Medical University, Beijing ChaoYang Hospital, Department of Stomatology, Beijing, China. 3 Sichuan University, West China School of Stomatology, State Key Laboratory of Oral Diseases, Chengdu, Sichuan, China.

J Appl Oral Sci.

90 2017;25(1):90-100

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

Introduction

derivatives. In the present study, spleen B cells from P. gingivalis non-immunized and immunized mice were

IL-10 expressing regulatory B cells (B10) is a VSHFL¿F ,/ FRPSHWHQW UHJXODWRU\ % FHOO VXEVHW WKDW KDV EHHQ UHFHQWO\ LGHQWL¿HG LQ ERWK PLFH DQG humans 27. B10 cell down-regulates autoimmune GLVHDVHLQÀDPPDWLRQDQGLPPXQHUHVSRQVHVWKURXJK IL-10 expression, playing crucial regulatory roles in innate and adaptive immunity27. Though mouse B10

co-stimulated with TLR4, TLR9, and CD40 signals to investigate their effects on B10 cell expansion and IL-10 competency in vitro.

Material and Methods P. gingivalisFXOWXUHDQG¿[DWLRQ

FHOOVVKDUHVRPHRYHUODSSLQJSKHQRW\SLFPDUNHUVZLWK

P. gingivalis (strain ATCC 33277) were grown on

RWKHUPXOWLSOHSKHQRW\SLFDOO\GH¿QHG%FHOOVXEVHWV

anaerobic blood agar plates (NHK agar, Northeast

they have been found to be predominantly enriched

Laboratory, Waterville, ME, U.S.A.) in an anaerobic

in spleen CD1dhighCD5+ B cells27.

chamber with 85% N2, 5% H2, and 10% CO2. Single

7ROOOLNHUHFHSWRUV7/5V ZKLFKEHORQJWRSDWWHUQ

colony of P. gingivalis was isolated from the plate

recognition receptors, are specialized transmembrane

and grown in ATCC Medium 2722. After incubation at

proteins that mediate innate immunity through

37°C for 4 d, bacteria number in culture medium was

detecting common structures of many microbial

determined by reading optical density values using

species such as bacterial lipopolysaccharides (LPS)

spectrophotometer and comparing them with a curve

. Upon recognition of a

derived from a standard plate count. Bacteria were

or viral nucleic acids

17,25

pathogen, TLRs initiate a signaling cascade that

FROOHFWHGDQG¿[HGZLWKSDUDIRUPDOGHK\GH3)$ 

OHDGVWRH[SUHVVLRQDQGUHOHDVHRISURLQÀDPPDWRU\

for 30 min at room temperature, then washed three

.

times with sterile phosphate-buffered saline (PBS) and

Porphyromonas gingivalis (P. gingivalis) LPS has been

resuspended in PBS at the concentration of 5×108/mL.

F\WRNLQHV FKHPRNLQHV DQG 7\SH, LQWHUIHURQV

8,21

shown to be able to activate both TLR2 and TLR4 due to its unique structure and function2,6, and CpG LV NQRZQ DV 7/5 DJRQLVW WR VWLPXODWH WKH LPPXQH responses

Animals &%/-PLFH-DFNVRQ/DERUDWRU\%DU+DUERU 0( 86$  DJLQJ  ZHHNV ZHUH HTXDOO\ DQG

.

9,16

Interaction between CD40 Ligand (CD40L)

randomly divided into four groups. Group 1 and 2

and CD40 plays an important role in the initiation

were set as non-immunized mice groups in which

and progression of cellular and humoral adaptive

PLFHZHUHVDFUL¿FHGGLUHFWO\IRUVSOHHQ%FHOOLVRODWLRQ

immunity15. The activation of CD40 on B cells by CD40L

Group 3 and 4 were set as immunized mice groups

is crucial for T cell-dependent B cell proliferation,

and mice were immunized by 1×108¿[HGP. gingivalis

differentiation, and antibody isotype switching11,13,14.

intraperitoneal injection at day 0, then followed by

Recent studies demonstrated that culturing

1×107¿[HGP. gingivalis injection at day 7 to enhance

spleen B cells with LPS or CD40L for 48 h induced

WKH LPPXQL]DWLRQ 0LFH ZHUH VDFUL¿FHG IRU % FHOO

VLJQL¿FDQWO\ KLJKHU IUHTXHQFLHV RI F\WRSODVPLF ,/

isolation at day 10. All mice used in the study were

10 production in B cells than control in vitro20. LPS

maintained under pathogen-free conditions in laminar

stimulation of spleen B cells for 24 h induced more

ÀRZFDELQHWV([SHULPHQWDOSURWRFROVZHUHDSSURYHG

IL-10 than unstimulated cells28. Spleen B cells with a

by the Institutional Animal Care and Use Committee

CD1d

CD21 CD23 MZ phenotype can produce IL-10

high

+

-

LQUHVSRQVHWR&S*VWLPXODWLRQLQPLFHZLWKOXSXVOLNH autoimmune disease30. However, despite all these

of the Forsyth Institute.

B cell isolation

¿QGLQJV WKH HIIHFWV RI 7/5 DJRQLVWV DORQJ ZLWK FR

Mice were euthanized in CO2 chamber and spleens

stimulatory molecules, such as CD40L, on B10 activity

were harvested. Single splenic cells were yielded by

during innate and adaptive immune responses are

JULQGHGRQDVWHHOPHVKDQGWKHQ¿OWHUHGZLWK

not clearly understood. Furthermore, there is limited

NjP &HOO 6WUDLQHUV $IWHU UHG EORRG FHOOV UHPRYDO E\

information on the role of B10 cells during immune

Ammonium-Chloride-Potassium (ACK) lysis buffer

responses to oral diseases, such as periodontal

(Life Technologies, Carlsbad, CA, USA), splenic cells

disease, when encountering oral pathogens and their

ZHUH UHVXVSHQGHG LQ 3%6 DQG ¿OWHUHG ZLWK  NjP

J Appl Oral Sci.

91 2017;25(1):90-100

LIU Z, HU Y, YU P, LIN M, HUANG G, KAWAI T, TAUBMAN M, WANG Z, HAN X

Cell Strainers. Then non-B cells were magnetically

(BioLegend, San Diego, CA, USA) on ice for 30 m using

ODEHOHGXVLQJ3DQ%FHOOLVRODWLRQNLW0LOWHQ\L%LRWHF

predetermined optimal concentrations. Then, all cells

&DPEULGJH 0$ 86$  %ULHÀ\ VLQJOH VSOHQLF FHOO

ZHUH FRXQWHG E\ ÀRZ F\WRPHWHUV %' %LRVFLHQFHV

suspensions were incubated with biotin-conjugated

San Jose, CA, USA) and data were analyzed by

monoclonal antibodies against non-B cell surface

FlowJo v10 software. For each sample, the same gate

PDUNHUV &' &'F &'E &' *U DQG

was applied to the other samples to determine their

Ter119) at 4°C for 10 min followed by incubation

CD1dhighCD5+ B cell percentages. Since mouse B10

with magnetic microbeads conjugated anti-biotin

cells has been found to be predominantly enriched

antibodies at 4°C for 15 min. Magnetically labeled

in spleen CD1dhighCD5+ B cells, CD1dhighCD5+ B cell

cells were then depleted by passing through LD

percentage is considered as the proportional indicator

columns (Miltenyi Biotec, Cambridge, MA, USA) under

of B10 cell percentage.

WKH PDJQHWLF ¿HOG RI WKH 4XDGUR0$&6Œ 6HSDUDWRU (Miltenyi Biotec, Cambridge, MA, USA). Unlabeled cells that passed through LD column were collected

IL-10 mRNA expression measurement 7RWDO P51$ RI % FHOOV ZDV LVRODWHG E\ 3XUH/LQN RNA Mini Kit (Life Technologies, Carlsbad, CA, USA)

(contained >98.5% CD19+ cells).

following the manufacturer’s instructions. Isolated

B cell culture

mRNA was then reverse transcribed to cDNA using

B cell number was counted by hemacytometer.

the SuperScript™ II Reverse Transcriptase system

Each 1×10  % FHOOV ZHUH FXOWXUHG LQ  Nj/

(Invitrogen, San Diego, CA, USA) in the presence

IMDM+GlutaMAXTM (Life Technologies, Carlsbad, CA,

of random primers following the manufacturer’s

USA) complete medium (contains 10% FCS, 100 U/mL

instructions. Then, real-time quantitative PCR (RT-

penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine,

T3&5  ZDV FDUULHG RXW LQ D  Nj/ UHDFWLRQ V\VWHP

NjJP/$PSKRWHULFLQ%DQGNj00( LQZHOO

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