MINIREVIEW

Microbiological Aspects and Immunity Response of Bacteria Causing Pulpitis

Deepak Dwivedi1, Sapna Kushwah1, Vinod Singh1
1

Department of Microbiology, Barkatullah University, Hoshangabad Road, Bhopal, M.P., India

Abstract
Oral cavity is a very wide distributed ecosystem in which several hundred microbial species normally cohabit harmoniously. Under special conditions some micro-organism with a potential is promoted, leading to inflammation & infection by induced demineralization of dental enamel that normally constitutes an impermeable barrier that protects the underlying dentin and the connective tissue situated in the centre of the tooth & dental pulp such as pulpitis, dental caries, periodontal infection. In inflammation process in the dental pulpitis the immunity response against oral infection follow leading infection & it is resulting in high level of morbidity and economic burden to society.

Nature Precedings : doi:10.1038/npre.2011.5889.1 : Posted 5 Apr 2011

Introduction
The human tooth is the target of a substantial number of oral bacteria agents that are responsible for the development of oral inflammation. These agents induce demineralization of enamel that normally constitutes an impermeable barrier that protects the underlying dentin and the connective tissue situated in the centre of the tooth, and dental pulp ( Love and Jenkinson 2002 ). When the enamel barrier is disrupted the dentin exposed to the oral environment. The pulpitis tissue is mainly composed of cells, loose connective tissue and ground substances. The main components are fibroblast, odontoblast, undifferentiated cells and defence cells. Dental pulp is also consists of nerves and blood supply
Corresponding Author Dr. Vinod Singh Asso. Professor & Head, Department of Microbiology, Barkatullah University, Hoshangabad Road, Bhopal, M.P., India, Phone no. +91755-4242852, Mobile No.-+919425712910 E-mail: vsingh3@rediffmail.com

Figure-1, Structural overview of Pulp Invasion of the pulp and the periapical areas can promote the development of pulpitis and dentoalveolar abscess and spread of the infection to other anatomical areas. Pulpitis can occur when caries progresses deeply into the dentin, when a tooth requires multiple invasive procedures, or when trauma disrupts the lymphatic and blood supply to the pulp. Untreated pulpitis may lead to pulp necrosis,

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Obligate anaerobic bacteria Genus Gram negative rods Porphyromonas prevotella Fusobacterium Propionibacterium Lactobicillus Actinomyces
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Common species P. gigivalis P.endodolis

P.oralis, P.oris, P.intermidius E.alactolyticum, E.lentum, P.propionicus L.catenaforme etc. A.naeslundii etc. P. anaerobius V.parvula

Figure-2, Mechanism of inflammation in pulp associated with the root canal infection finally lead to apical periodontitis, destruction of the bone surrounding the root apex of affected tooth. Pulpitis begins as a reversible condition in which the tooth can be saved by a simple filling. It becomes irreversible as swelling inside the rigid encasement of the dentin compromises circulation Several oral acid producing aerobic and anaerobic bacteria. There is a chamber in the centre portion of each tooth, and that chamber contains a mix of nerves, blood vessels and soft, spongy tissue called pulp. Healthy pulp causes no sensations, but when it is attacked by bacteria, pressure builds up in the chamber and you experience pain. Pulpitis is typically caused by untreated caries (decay) that has inched its way down into the pulp chamber. Your simple toothache could have also been the result of a loose filling, periodontal disease, an injury to the tooth or an extensive, invasive dental procedure that came close to the pulp chamber.

Gram positive cocci Gram negative cocci

Peptostreptococcus Veillonella

Facultative anaerobic bacteria Gram positive cocci streptococcus Enterococcus S.mitis, S.oralis etc. E.faecalis

Gram Neisseria negative cocci Gram cornibacterium positive rod Gram Eikenella negative rod The interaction between micro flora and eukaryotic cell is highly complex and involves active processes allowing both type of partner to co-exist. The microbes encounter with the host and bacterial surface protein termed adhesions mediate this step. Bacteria also secret mediators that bind or invade the host, he microbial attachment as well as the encounter with the secreted protein serve as signals that are deciphered through multi mediator cascades ultimately affecting gene expression in the host. Many receptors recognizing the pathogens and mediating the host response, as well as the variety

Predominant isolated bacteria from necrotic infected pulp

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of microbial molecular triggering the host response have been demonstrated.

Toll like receptor and bacteria present in pulpitis

Host defence against invading microbial pathogens is elicited by the immune system, which consists of two components: innate immunity and acquired immunity. Both components of immunity recognize invading microorganisms as non-self, which triggers immune responses to eliminate them As above described in caries causing pulpitis both gram positive and gram negative bcteria are present and TLRs function as signal transducers that mediate innate immunity and inflammatory response to pathogen through pattern recognition of virulence molecules. Mammalian innate immune system recognizes bacteria and their cell wall components through and pattern recognition receptors CD14 and TLR 2and 4, Although the exact mechanism of immune activation by Grampositive bacteria remains unknown, recent studies of immune activation by bacterial LPS provide a clue. TLR2 recognizes a variety of microbial components. These include lipoproteins/lipopeptides from various pathogens, peptidoglycan and lipoteichoic acid from Grampositive bacteria. Whereas TLR4 seems to serve as the primer LPS receptors. Some researchers have shown that TLR 2 may not be exclusively pattern recognition receptors for LPS from Gram negative bacteria (Schwandner et al 1999). To study the expression of TLRs by odontoblasts, we used a pure population of cells generated in vitro that are similar in many aspects to in vivo odontoblasts ( Couble et al 2000,Lucchini et al 2002).We observed the constitutive expression of TLR16 and 9 genes but not TLR7, 8, and 10 genes. This large range of TLRs expressed by odontoblasts appears comparable to what has been reported for cultured epithelial cells, including

keratinocytes intestinal epithelial cells , bronchial epithelial cells (sha et al 2004)and gingival epithelial cells . Interestingly, the pattern of TLRs expressed by odontoblasts was similar to the one reported for gingival fibroblasts in primary cultures . Whether a common TLR expression profile exists for all oral mesenchymal cell types remains to be determined. Thus, odontoblasts might be involved in the recognition of bacterial products such as triacetylated lipoproteins (TLR1+TLR2), LTA (TLR2), diacetylated lipoproteins, peptidoglycans (TLR2+TLR6), LPS (TLR4), flagellin (TLR5), and unmethylated CpG motif-containing DNA (TLR9), and also of viral dsRNA through TLR3 (Takeda et al 2002, Iwasaki 2004). Indeed, we found in the present work that odontoblasts responded in vitro to the TLR2 ligand LTA but also to TLR3 and TLR4 ligands (our preliminary data). It remains to be determined whether these cells can actually detect and react to TLR5 and 9 ligands. Although the dental pulp is equipped with cells of the immune system (Jontell 1986), the immune response in the pulp to caries pathogens is poorly understood. The basis for the earliest step in innate immune response to Gram positive bacterial infection is poorly understood. We hypothesized that Gram positive bacteria might also be recognized by TLRs. Although the exact mechanism of immune activation of Gram positive bacteria remains unknown, recent studies of immune activation by bacterial LPS provide a clue. Like Gram negative bacteria, major component of the gram positive bacterial cell wall employ CD14 for immune recognition. Both peptidoglycan and lipoteichoic acid have been demonstrated to activate macrophages in a CD14-dependent manner (Gupta 1996)

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Gram positive bacteria ent G e tering the den ntinal tissue during the car process ar suspected t influence d ries re to the immune response in human de n ental pulp. Odontoblasts situated at th pulp are th first cells O he he encountered by these bacteria and ther e b refore could play a crucial role in this re p esponse. The predomin T nant role of G Gram-positive bacteria in e dental caries, we analyzed further the odontoblast d d response to L r LTA. LTA wa found to strongly upas s regulate the e r expression of its own recep ptor, TLR2, and to a lesse extent, of T a er TLR3, 5, and 9. The upd regulation of TLR2 by its ligand may i r increase the sensitivity of odontoblasts as previous reported s s, sly in hemopoieti cells (Ray e al 2003). T absence ic et The of TLR2 de o etection in resting odon ntoblasts is probably beca p ause the prot tein is presen at a level nt below the sen b nsitivity threshold of flow cytometry, as reported for iDCs (Visin et al 2001 a ntin 1).

Immu unity agains pulpits ca st ausing bacte eria

Pulpiti is characte is erized as the immune res sponse that is mainly trigg gered by the invasion of c cariesrelated micro-organ d nisms into de entinal tubule and es pulp. The i innate immu une response associated with e infecti ions is link ked to the adaptive im mmune respon by cytokin production nse ne n. Cytoki inesproductio in pulp by bacteria -The on lesions in dental pu under sha s ulp allow caries a Tare cell do ominated, wit CD8+ T ce predomin th ells nating. As car progresse and the deep lesion em ries es merges, + the CD T cell con D8 ntinues to dom minate, but CD + T D4 cells, B cells, a and plasma cells appear in substan ntial number (Hahn et al 1989;Izumi et al rs i 1995). A fea ature of pul lpal immune responses i the is predom minance of t type 1 cytok kine mRNA under shallow caries and a mixed (typ 1/type 2) p w pe profile under deep caries These res s. sults prompte an ed examin nation of the cytokine pr e rofiles induced by bacteri in shallow caries (Stre ia w eptococcus m mutans and Actinomyces viscosus) and deep caries A (Lacto obacillus casei, Pseudoramib bacter alactolyticus, and Prevotella intermedia) All ). isolate induced int es terferon- and interleukind -10, S. mutans induced s s substantially more interfe eron- than interleukin-10 suggestin strong ty 0, ng ype 1 polariz zation. P. ala actolyticus ind duced significantly more interleukin-10 than interf feron-, sugg gesting polariz zation toward type 2. The high titers of IL-12 d h induction by S.muta agree wit previous studies ans th using different t S.mutan ns s strains (Jiang, ,Magli&Russ so 1999, Plitnick e et al 1998).Hahn,Best & tew,20 000 first time demon nstrated th he presence e of mu ultiple inflam mmatory cytok kine mRNAs (IFN- , IL-4 and 4, IL-10) in the human dental pulp in carious les ) n sions. It is known that T-cells are most likel the t e ly predom minant lymph hocyte popul lation in inf flamed dental pulp tissue (Hahn et al 1989;Izumi et al l

Nature Precedings : doi:10.1038/npre.2011.5889.1 : Posted 5 Apr 2011

Figure-3 Role of TLRs in oral Inflamm F e mation


1995). Chem 1 mokines are responsible for the e recruitment an subsequen activation of particular r nd nt o leukocytes, su as activat T-cells, in inflamed uch ted nto tissues via spe ecific chemok kine receptor expressed rs on the cells (S o Sallusto et al 2000); howe ever, little is known about the role of chemokin k t nes in the Increased ex I xpressions of chemokine such as f es interleukin-8, C-C chemo okine ligand (CCL) 20, and CCL2 a found in inflamed dental pulp a are n d (Huang et al 1999,Nakans et al 2005 ( shi 5,Durand et al 2006); resu indicate th CCL20 ex a ults hat xpression is induced by sti imulation wit caries-relat bacteria th ted that have inva aded deeply i into the denti inal tubules as well as by proinflamm a y matory cytok kines in the inflamed pulp lesions. It may be invo pal t olved in the progression of pulpitis via accum p mulation of inflammatory cells. howe ever, the mec chanism of activated lym a mphocyte infi filtration in d dental pulp tissues was unclear. In pe eriapical granulomas, the presence of other chemo p okines (such as CCL3, CCL4, and CCL5) re C elated to l lymphocyte recruitment h been dem r has monstrated (M Marton et al 2000,Kabashi 2 imo et al 20 001). These c chemokines might contrib m bute to the formation of chemokine f networks in ac n ctivated T-cell infiltration in inflamed dental pulp l d lesions, altho ough they ha not yet ave been elucidat b ted. In the p present study we first y, demonstrated that CXCL10 expression in inflamed d 0 dental pulp tissues was significantly increased d y compared wi that in healthy denta pulp. In c ith h al addition, man CXCR3-e a ny expressing T-cells were observed in in o nflamed pulp. These findin suggest ngs that CXCL10 may act as a key chemo 0 okine in the accumulation of activated lymphocytes in pulpitis. a accumulation of lymphocy into the dental pulp a ytes lesion. Bacteria can initiate cellB -mediated im mmunity by stimulating macrophages to produce int s o terleukin-12 (IL-12), which can then pro ( h omote gamma interferon (IFN- ) produ ( uction by nat tural killer ce ells. IFN- activated mac a crophages the secrete mo IL-12 as en ore a result of positive feed p dback, leadin to cellng mediat ted Th1 res sponses. IFN N- also act tivates macrop phages to p produce tumo necrosis factor or alpha (TNF- ), w which in tu urn maintain the ns activat state of t macropha (samaran ted the age nayake 1996). TNFis a potent proinflamm s matory cytokin that can s ne stimulate chem mokine produ uction by en ndothelial ce ells and fib broblasts in vitro (Sylve ester et al 199 In additio to its prot 90). on tective role in the host def n fence against infectious a t agents, TNF- contributes to bone reso orptive activit and ty has be implicated in patholog bone resorption een d gic (Pfizen nmaiener et a 1993, Stas al shenko et al 1987, Thomp pson, Mundy, Chambers 1987). An eff 1 fective host defense ag gainst bacterial invasio on is charac cterized by the vigorous recruitmen and s nt activat tion of inf flammatory cells, which are h modul lated by the c coordinated expression of both e f pro- and anti-inf flammatory cytokines. I IL-10, which is produce by mon ed nocytes and Th2 lymph hocyte subset inhibits IF ts, FN- synthes by sis Th1 cells and inhibits the productio of on proinfl flammatory c cytokines such as IL-12 and 2 TNF- . The inhibit effects of IL-10 on cyt ting f tokine produc ction correlat with their antiinflamm te r matory effects in vivo (L Elliott & Monsmann1 s Li, 1994). Theref fore, IL-10, I IFN- , IL-12 and TNF- have 2, import tant and cros ss-regulatory roles in inf fection (Trinch hieri 1997). It is k known that im mmune cells in inflamed dental pulp produce a va p ariety of cyto okines, which can modify the pathoge y enesis of pul lpitis.Streptoc coccus mutans elicite mu ultiple inflam mmatory cyto okines IFN y , IL- 4, IL-10 are detecte in human dental y ed pulp ( Hahn et al 2000). Lipo l oteichoic acid and peptidoglycan are know to in nduce IL 1 by 12 dendri cells or m itic monocytes an monocyte and nd es IL-12 promotes ind ductions of T 1 response and Th es IFN-Y production ( Rissoan et a 1999) Y al P. ging givalis posses sses bioactive materials su as e uch cytoplasmic memb branes, pept tidoglycans, outer membr rane protein ns, lipopolysaccharide ( (LPS), fimbriae cell capsul les, and on their

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surface(Offen s nbacher,1996) These mater ). rials induce excessive pr e roduction of cytokines and may f modulate the cytokine n m e network in periodontal tissues ( Genc and Slots, 1984). Severa cytokines co al are involved in inflam a d mmatory as well as immunologica responses, and are designated as al inflammatory cytokines. P. gingivalis LPS enhan P nces the pro oduction of inflammatory cytokines su as interleu uch ukin (IL)-1, IL-6, IL-8, an tumor necr I nd rosis factor a alpha (TNF).Marked inf filtration of inflammatory cells, such i as activated T a T-cells, is observed in the p progression of pulpitis; B o Both S. mutan and L. pla ns antarum are Gram-positive bacteria, which have ba G e w acterial cell wall components such as LTA and PGN. It is w s known that LT and PGN are recognize by TLR2 k TA ed (Michelesen et al 20 ( 001). Adachi et al 2007examined 2 d,HDPF (h human den ntal pulp fibroblasts) st f timulated wi PGN, but not LTA, ith t were able to p w produce CXCL L10. The macropha appears t be a key ce involved T age to ell in host resp ponse to LP PS. After release from bacteria, LPS is initially bo b ound to a plas sma protein called LPS-b c binding prote (LBP) an is then ein nd delivered to C d CD14, a cell receptor for LPS on the surface of ma s acrophages. S Subsequent ac ctivation of the macrophage is a result of signal trig t ggered by a signal-transud s ding receptor called Toll-li receptor ike (TLR). The T family of receptors en ( Toll f ncompasses trans membran molecules linking the e ne extracellular compartment, where cont c tact and reco ognition of pathogens p occurs, an nd the intracellular i compartment, where signal c lling cascades leading to cellular resp c ponses are initiated. TLRs are responsible f cell sign r for nalling to a variety of bacterial com b mponents. TLR-4 is in T nvolved in cellular activ c vation by LP from most bacteria. PS However, TL H LR-2 may be involve in cell ed signalling to s s some types of LPS, such a that from f as Porphyromon P nas gingivali Engageme of the is. ent receptor acti r ivates transc cription facto ors, which induce activation of genes encoding several e cytokin nes. Ackno owledgment
Author would like to acknowle rs edge Department of Microb biology Barkatullah University, al, Bhopa & Peoples Dental Acade emy for necess sary help provi ided to ct nd upport by a gran from nt conduc the study An Financial su
UGC (U University Grant Commission) N Delhi, India t New a.

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