Intracellular and extracellular C. pneumoniae immunoreactivity was observed in the entorhinal cortex, the hippocampal formation, and the frontal cortex, regions of the brain that typically demonstrate AD pathology. Clear discrimination between chlamydia immunoreactivity and age-related lipofuscin accumulation within neurons was demonstrated. Serial sections of brain tissue displayed both amyloid pathology and the presence of C. pneumoniae immunoreactivity. Thioflavin S staining for fibrillar amyloid and specific antibody labeling for C. pneumoniae revealed deposition of both when performed on the same section. As some C. pneumoniae labeling was extracellular, a more atypical pattern, pre-absorption studies with Amyloid β 1-40 and 1-42 were performed. These studies revealed that C. pneumoniae antibodies were not cross-reacting with Aβ. Collectively, these data demonstrate that evidence of C. pneumoniae infection is present in brain tissues in areas of amyloid pathology, thereby suggesting that an interrelationship exists between these entities in the pathogenesis of sporadic late-onset AD.
Immunolabeling for Chlamydia may be overlooked in brain tissues, as it is different from what is observed in cellular infections in vitro. In all AD samples analyzed in this study, both typical intracellular perinuclear chlamydia immunoreactivity and atypical extracellular labeling were observed. Intracellular labeling demonstrated punctate elementary bodies and membrane bound inclusions similar to that of in vitro studies (see Figure ). This specific labeling was differentiated from lipofuscin by using red chromogens, either alkaline phosphatase (AP)red or AP magenta, as the substrate to denote C. pneumoniae immunoreactivity. Horseradish peroxidase labeling with 3, 3′-Diaminobenzidine (DAB), a brown chromogen, was not used as this labeling may be confused with the golden brown lipofuscin found in neurons of aged brains.
Two distinct extracellular patterns of chlamydia immunoreactivity were observed: one, a punctate pattern signifying the elementary body form of the bacteria, which can be extruded from infected cells into the surrounding milieu [31
], and two, an amorphous foci pattern most likely indicating secreted chlamydial factors such as lipopolysaccharide [30
]. These patterns in the cerebrum will require further study although similar profiles of Chlamydia labeling in situ
have been demonstrated in a different organ [32
]. Furthermore, our data demonstrated that C. pneumoniae
extracellular immunoreactivity was not reflective of cross-reactivity with extracellular Amyloid β 1-40 or 1-42. However, C. pneumoniae
extracellular organism and related antigens may interact with extracellular proteins and lipids in the brain. Although not always in direct overlap with amyloid plaque deposits, chlamydial antigens may interact with soluble oligomeric forms of amyloid, such as ADDLs, that are less likely to be found in mature plaques due to their soluble nature [7
]. These intriguing findings and their implications require further understanding of the possible relationship between amyloid and chlamydia in the same cortical regions of the brain. This relationship will vary with each individual AD patient. Each AD patient has different levels of pathology and may have corresponding variability in extent and distribution of C. pneumoniae
infection in the cerebrum. Following further studies into this variability, the relationship between pathology and infection can be more thoroughly evaluated.
Although C. pneumoniae
is principally a respiratory pathogen, infection of the brain has been shown following intranasal and lung infection [33
]. In this regard, monocytes infected with C. pneumoniae
in the lungs may spread the infection via the peripheral circulation to the brain through the blood brain barrier or circumventricular organs [22
]. Alternatively, a more direct and insidious route of infection may follow the olfactory pathways. As such, the infection becomes established in the olfactory nasal neuroepithelia, progresses to the olfactory bulbs, and eventually infects brain structures such as the entorhinal cortex and hippocampus. The olfactory structures, the entorhinal cortex, and the hippocampal formation are the most vulnerable and the earliest regions affected in the onset of AD [35
]. Our current study highlights C. pneumoniae
detection in the frontal and temporal cortices, including the entorhinal cortex and the hippocampal formation. Thus, infection of these regions in the brain may have great impact on the development of AD pathology.
Previous studies have demonstrated C. pneumoniae
in both human and animal olfactory bulbs [8
]. In both cell culture and animal studies, C. pneumoniae
has been shown to infect nasal neuroepithelial cells [34
]. In the animal studies, infection appeared to spread centrifugally from the vulnerable neuronal cells in the olfactory bulb into the brain [33
]. Further, Chlamydia isolated from AD brains in a prior study was shown to have more commonalities with Chlamydia respiratory strains than with Chlamydia strains from atherosclerosis with the suggestion that the organism itself may have a tropism for specific cell types in the CNS [38
]. Upon consideration of these data and our current data, a rationale for the selective vulnerability of specific brain regions to infection and resultant pathology emerges.
Notably, histopathological amyloid plaques and tangles are used to define the stage of AD, but the correlation with the pathology of the disease and the clinical manifestations of the disease are not always clear [2
]. Some individuals who have massive pathology have little to no symptoms. On the other hand, some symptomatic individuals may show little pathology upon post-mortem histopathological examination. As such, there are many variations in the amount and type of damage evident in AD [1
]. The variability of correlation between the symptomology and histopathology suggests other events and/or ingredients may be missing in the pathobiology of AD.
The response in the brain to infection may determine the extent of pathology and symptomatology that may arise. In this regard, C. pneumoniae
infection characteristically promotes an inflammatory response whereby cytokines such as IL-1β and TNF-α are secreted and may initiate cellular damage [39
]. These cytokine responses to infection parallel similar responses to amyloid accumulation [40
]. Additionally, another cellular response to C. pneumoniae
infection in culture is the production and processing of amyloid peptides. Labeling of infected cells in culture for Amyloid β 1-42 often reveals intracellular immunoreactivity at early post-infection times (unpublished observations CJH, DMA, CSL, BJB). Interestingly, our earlier study demonstrated intracellular and extracellular amyloid deposits in the brains of non-transgenic BALB/c mice following intranasal C. pneumoniae
]. Our current study showed a similar relationship in which we demonstrated both amyloid and chlamydia immunoreactivity in the temporal cortex of the AD brain.
Further evaluation is required to specifically demonstrate how amyloid and C. pneumoniae
, both intracellular and extracellular, are interrelated. In addition, future studies are required to further characterize the atypical extracellular Chlamydial immunolabeling profiles. Others have demonstrated evidence for an association of infection and amyloid in AD [41
]. HSV1 viral DNA was shown to specifically associate with AD amyloid plaques [41
]. Previous hypotheses have even suggested that amyloid in the AD brain may act as an antiviral agent [42
] or an entrapping agent for infection [43
]. Intriguingly, a recent study suggests that amyloid has anti-microbial properties, and may arise in response to brain infection in AD [44
]. As 2 of 5 non-AD cases in our current study showed occasional chlamydia immunoreactivity and diffuse amyloid deposition, future analysis must also include mild cognitive impairment cases, as well as non-AD cases, as infection may be a prodromal event leading to eventual AD pathology.