group b streptococci ( gbs ) have long been known as a leading cause of life - threatening infection in neonates , young infants , and pregnant women [ 13 ] . recently these microorganisms have been recognized as an ever - growing cause of serious invasive infections in nonpregnant adults [ 46 ] . the most common entities include primary bacteraemia , skin and soft tissue infections , urinary tract infections , and pneumonia . other relevant conditions are endocarditis , intravascular device infections , meningitis , peritonitis , endoophthalmitis and osteoarticular infections . bacterial arthritis is a rapidly progressive and highly destructive joint disease in humans   with an incidence rate ranging from 0.034% to 0.13% . the typical case of this disease is characterized by high fever and a red , warm , and swollen joint , in 80% to 90% of cases displaying a monoarticular pattern . patients with primary or secondary   immunodeficiencies as well as those who have passed through prosthetic joint surgery display enhanced susceptibility to septic arthritis [ 1012 ] . septic gbs arthritis in nonpregnant adults was considered extremely rare [ 13 , 14 ] until the early 1980s , when two independent studies stressed that its incidence seemed to be increasing [ 15 , 16 ] . this trend has been confirmed by recent studies in which gbs account for 7% to 10% of all diagnosed cases of bacterial arthritis [ 17 , 18 ] . interestingly , gbs were typically associated with involvement of multiple joints in patients with diabetes mellitus or cancer . we have previously described an experimental mouse model of type iv gbs infection with clinical features that closely resemble infection in humans [ 19 , 20 ] . mice given a single intravenous dose of gbs develop clinical signs of arthritis within 48 hours . appearance and severity of gbs arthritis are the by - product of a multifactorial process . viability and number of microorganisms injected and bacterial factors ( i.e. , presence and amount of capsule , amount of sialic acid in the capsular polysaccharide , and   -hemolysin production ) have been shown to influence the development of articular lesions [ 21 , 22 ] . nevertheless , in the pathogenesis of gbs arthritis a crucial role is played by inflammatory cells ( granulocytes and monocytes ) that reach the joints [ 23 , 24 ] and by the production of proinflammatory cytokines , including interleukin-6 ( il-6 ) il-1 and tumor necrosis factor- ( tnf- ) . il-4 suppresses proinflammatory cytokines ( tnf- il-1 , and il-12 ) [ 26 , 27 ] and chemokines ( interferon--inducible protein 10 and macrophage migration inhibitory factor ) [ 28 , 29 ] . it has been demonstrated that systemic administration of il-4 effectively suppresses disease in proteoglycan - induced arthritis ( pgia ) and collagen - induced arthritis ( cia ) [ 30 , 31 ] . treatment with il-4 or local expression of il-4 in cia prevents cartilage and bone destruction [ 32 , 33 ] . the aim of our study was to assess the role of il-4 in our septic arthritis model , both regarding development of the arthritic process and host susceptibility to bacteria . for this purpose , we used balb / c wild - type mice and balb / c mice with disrupted il-4 gene . homozygotous il-4/ mice , generated as described elsewhere , have been kindly provided by prof . control wt balb / c   mice were purchased from charles river breeding laboratories ( calco , italy ) . the animals were maintained and used under strict ethical conditions according to recommendations of european union 's directive no . type iv gbs , atcc reference strain gbs 3139 ( cnctc 1/82 ) , supplied by j. jelinkova ( prague , czech republic ) was used throughout the study . for experimental infection , the microorganisms were grown overnight at 37c in todd - hewitt broth ( oxoid ltd . , basingstoke , hampshire , england ) and then washed and diluted in rpmi 1640 medium ( gibco , life technologies , milan , italy ) . the inoculum size was estimated turbidimetrically , and viability counts were performed by plating on tryptic soy agar   5% sheep blood agar ( blood agar ) , and overnight incubation under anaerobic conditions at 37c . a bacterial suspension was prepared in rpmi 1640 medium . mice were inoculated intravenously via the tail vein with 1      10 gbs in a volume of 0.5 ml . control mice were injected by the same route with 0.5 ml of rpmi 1640 medium . after challenge , mice were examined daily by two independent observers ( l. t. , m. p. ) for 30 days to evaluate the presence of joint inflammation , and scores for arthritis severity ( macroscopic score ) were given as previously described [ 26 , 35 ] . clinical severity of arthritis was graded on a scale of 03 for each paw , according to changes in erythema and swelling ( 0 = no change ; 1 point = mild swelling and/or erythema ; 2 points = moderate swelling and erythema ; 3 points = marked swelling , erythema , and/or ankylosis ) . the arthritis index ( mean      sd ) was constructed by dividing the total score ( cumulative value of all paws ) by the number of animals used in each experimental group . gbs - infected mice were examined 10 days after infection for histopathological features of arthritis . arthritic hind paws ( 1 per mouse ) were removed aseptically , fixed in formalin 10% v / v for 24 hours and then decalcified in trichloroacetic acid 5% v / v for 7 days , dehydrated , embedded in paraffin , sectioned at 3 - 4 m , and stained with hematoxylin and eosin . tibia - tarseal , tarsus - metatarseal , and metatarsus - phalangeal joints were examined , and a histologic score was assigned to each joint based on the extent of infiltrate ( presence of inflammatory cells in the subcutaneous and/or periarticular tissues ) , exudate ( presence of inflammatory cells in the articular cavity ) , cartilage damage , bone erosion , and loss of joint architecture . arthritis severity was classified as negative ( no infiltrate ) , mild ( minimal infiltrate ) , moderate ( presence of infiltrate , minimal exudate , and integrity of joint architecture ) , and severe ( presence of massive infiltrate / exudate , cartilage and bone erosion , and disrupted joint architecture ) . blood , kidney and joint bacterial load in gbs - infected mice was determined by colony - forming - units ( cfu ) evaluation at different times after inoculation . ten - fold dilutions were prepared in rpmi 1640 medium ,   and 0.1 ml of each dilution was plated in triplicate on blood agar and incubated under anaerobic conditions for 24 hours . the number of cfu was determined , and the results were expressed as the number of cfu per ml of blood . all wrist and ankle joints from each mouse were removed , weighed , and homogenized in toto in sterile rpmi 1640 medium ( 1 ml/100 mg of joint weight ) . after homogenization , all tissue samples were diluted and plated in triplicate on blood agar , and the results were expressed as the number of cfu per whole organ or per ml of joint homogenate . blood samples from the different experimental groups were obtained by retroorbital sinus bleeding at different times after infection before the mice were killed . briefly , all wrist and ankle joints from each mouse were removed and then homogenized in toto in 1 ml/100 mg joint weight of lysis medium ( rpmi 1640 containing 2 mm phenylmethylsulfonyl fluoride and 1 g / ml final concentration of aprotinin , leupeptin , and pepstatin a ) . the homogenized tissues were then centrifuged at 2000 g for 10 minutes , and supernatants were sterilized using a millipore filter ( 0.45 m ) and stored at   80c until analyzed . il-6 , il-1 ,   tnf- , macrophage inflammatory protein ( mip)-1 ,   and mip-2 concentrations in the biological samples were measured with commercial elisa kits purchased from r&d system ( r&d systems inc . the detection limits of the assays were 1.6 pg / ml for il-6 , 3 pg / ml for il-1 , 1.5 pg / ml for mip-1 1.5 pg / ml for mip-2 , and 5.1 pg / ml   for tnf-. differences in the arthritis index , number of cfu , and cytokine concentrations between the groups of mice were analyzed by student 's t - test . the log - rank test was used for data analyses of kaplan - meier survival curves . balb / c mice defective ( il-4/ ) or intact ( wt ) with respect to il-4 gene were injected i.v . with 1      10 gbs and the clinical course of disease the severity of the disease was monitored by assessing both survival rates and clinical score of arthritis . as shown in figure 1(a ) statistically significant ( p = .0372 ) differences were observed in mortality rates between the two experimental groups . in fact , infection with 10 gbs per mouse resulted in a 10% mortality in il-4/ mice , while 40% of wt mice had died at the end of observation period . the clinical signs of joint swelling were observed as early as 24 hours after injection of 1      10 cfu of gbs in both groups of mice . although similar frequency of articular lesions was observed in il-4/ and wt mice ( figure 1(b ) ) , il-4 deficient group had a more severe clinical arthritis in affected joints , compared with the control animals , resulting in a higher arthritis index ( figure 1(c ) ) . significant between - group differences were evident throughout the observation period , with maximum values reached at day 10 after infection of 3.2      0.5 in il-4/ mice versus 2.0      0.3 in wt animals . arthritic paws were removed for histological examination at day 10 after infection with 1      10 gbs per mouse and examined for histopathological features of arthritis . five arthritic paws from both il-4/   mice and balb / c   mice were removed , and 3 joints for each paw were assessed for histopathological score . as shown in figure 2 , none of the joints from control animals could be classified as severely affected . in this group , 39% of the joints were classified as mildly affected , 32% as moderately affected , and 29% did not show any sign of arthritis . in contrast , in il4/ mice only 5% of the joints were negative , while the majority of them were classified as moderately ( 49% ) or severely ( 31% ) affected . in vivo gbs growth was assessed 2 , 5 , and 10 days after gbs infection by quantitative monitoring of bactaeremia and bacterial growth in kidneys and joints of il-4/ or wt mice . as shown in figure 3 , a progressive clearance of bacteria was observed in the blood of wt as well as il-4/ animals . however , it is noteworthy that a significantly ( p < .05 ) lower number of gbs were recovered in the bloodstream of il-4/ mice in comparison to controls , as soon as   two days after infection . a lower streptococcal growth was also observed in the kidneys from il-4/ mice with respect to controls . ten - fold between - group differences in the number of gbs recovered were found at all the time points assessed . finally , as already assessed in the kidneys and in the blood , bacterial burden in the joints of   il-4/ mice a progressive bacterial growth   was then observed in the joints of both experimental groups , although a significantly lower   bacterial load was still present in il-4/ mice . the role played by cytokines and monokines on the pathogenesis of gbs sepsis and arthritis is well - defined [ 24 , 25 ] . thus , local and systemic levels of il-6 , il1 ,   tnf- , mip-1 , and mip-2 were compared between il-4/ and control mice at 2 , 5 , and 10 days after infection . as depicted in figure 4 , higher levels of proinflammatory cytokines ( il-6 , il1 and tnf- ) were detected in the joints of il-4/ mice than in controls . at day 2 after infection significantly higher levels were evident only with regard to tnf- production , but at the other time points assessed differences became statistically significant also for il-6 and il-1 production . on the contrary , serum levels of proinflammatory cytokines were similar in both groups , except for tnf- production which was significantly higher in il-4/ mice with respect to controls ( 52      10 versus 11      5 pg / ml of serum ) at day 2 after infection . a similar trend was observed for mip-2 and mip-1 production in both groups of mice , with peak values reached at day 5 after infection . again , significantly higher levels of both chemokines were detected in the joints of il-4/ mice than in wt animals at all the time points assessed ( figure 5 ) . differently from the joints , no significant differences were observed between the two experimental groups with respect to systemic production of mip-1 and mip-2 ( data not shown ) . the murine model of gbs infection has proven to be beneficial in elucidating bacterial and host factors responsible for gbs arthritis [ 1925 ] . our previous studies pointed out   a major role for proinflammatory cytokines such as of il-6 , il-1 ,   il-18 , and tnf-   in the pathogenesis of gbs arthritis [ 25 , 36 ] . in this study , by using deficient mice , we found that the antiinflammatory cytokine il-4 has a relevant influence on gbs - induced sepsis and septic arthritis . the first effect of il-4 absence on the course of gbs infection is the decrease of mortality rates . this result is in line with a paper by hultgren et al . , who described a beneficial effect of il-4 lack in an experimental murine model of staphylococcus aureus     infection . in this paper , the diminished mortality rates observed in il-4/ mice were associated to an increased systemic bacterial clearance , attributable to an enhanced intracellular killing capacity mediated by phagocytic cells . it has been demonstrated that il-4 acts on phagocytes by suppression of oxidative bursts and intracellular killing of leishmania     , and inhibits peritoneal and splenic macrophage killing of candida albicans    . actually , in our experimental model a lower number of gbs were recovered both from blood and kidneys of   il-4/ mice compared to controls , thus suggesting that il-4 exerts an inhibitory effect also on gbs killing by phagocytes . this was apparently in contrast with the lower bacterial load found in the joints of il-4/ mice with respect to controls . however , it must be considered that severity of gbs arthritis is not exclusively linked to the amount of bacteria present in the joints , but is also dictated by other parameters , including proinflammatory cytokine production and the extent of inflammatory infiltrate / exudates in the articular tissues . the recruitment and activation of   inflammatory cells at infection sites is a complex and dynamic process that involves the coordinated expression of both pro- and antiinflammatory mediators . indeed , although an inflammatory response is essential to clear pathogens from the site of infection , a prolonged inflammatory response might lead to a detrimental outcome . in this respect , a beneficial effect of il-4 has been demonstrated in many experimental model of arthritis . administration of exogenous il-4 resulted in suppression of cia , while , in the same experimental model , administration of anti - il4 monoclonal antibodies augmented both incidence and severity of arthritis . daily treatment with il-4 was reported to reduce all disease parameters in a streptococcal cell wall ( scw ) rat arthritis model . in the pgia model , arthritis was prevented by il-4 treatment   and exacerbated in il-4/ mice . in these mice , the rapid induction of arthritis parallels in vivo increase of proinflammatory cytokine and chemokine secretion . it is well known that proinflammatory cytokines and chemokines play a critical role in the pathogenesis of arthritis . resident synovial macrophages and fibroblasts as well as infiltrate cells produce proiflammatory cytokines , such as tnf- , il-1 , il-6 , il-15 , and il-17 that drive disease pathogenesis [ 42 , 43 ] . these cytokines initiate a cascade of events that induce cartilage - degrading enzymes , such as metalloproteases , and that stimulate osteoclastogenesis and angiogenesis [ 4446 ] . recruitment of cells to the synovial tissues involves chemokines , such as mip-1 responsible for recruitment of mononuclear cells and mip-2 that acts on neutrophils . all these inflammatory cells contribute to articular damage by cytokine and proteolytic enzyme production . in our model , despite the lower number of gbs in the joints of il-4/ mice , all the other parameters leading   to an exacerbation of articular lesions are manifested . in fact , worsening of joint disease was associated with a marked increase of local proinflammatory cytokines ( il-6 , il-1 ,   and tnf- ) responsible for articular damage , together with sustained levels of monokines and a consequent enhanced inflammatory cell influx . in conclusion , we propose that il-4 plays a dual role in the immune response to gbs infection . by one side , by inhibiting the intracellular killing of phagocytes , il-4 may cause a more severe systemic infection and , consequently , higher mortality rates . by the other side , by downregulating local levels of proinflammatory cytokines and chemokines , it appears to positively influence the progression of gbs arthritis .
il-4 is an 
                   anti - inflammatory cytokine that inhibits the 
                   onset and severity in different experimental 
                   arthritis models . 
 group b streptococci ( gbs ) 
                   have been recognized as an ever - growing cause of 
                   serious invasive infections in nonpregnant 
                   adults . 
 septic arthritis is a clinical 
                   manifestation of gbs infection . to investigate 
                   the role of il-4 in experimental gbs infection 
 , 
                   il-4 deficient or competent mice were inoculated 
                   with 1  107 gbs / mouse . 
 mortality , appearance of arthritis , gbs 
 growth in the organs , and local and systemic cytokine and 
 chemokine production were examined . 
 il-4/   mice 
 showed lower mortality rates but increased severity of arthritis 
 and exhibited a lower microbial load in blood , kidneys , and joints 
 than wt mice . increased local levels of il-1  , il-6 , tnf- , mip-1 , and mip-2 accompanied the more severe arthritis in il-4/ mice 
 . 
 our results suggest a detrimental role of il-4 in gbs sepsis , whereas it plays a beneficial effect on gbs - induced arthritis .