Discussion
Although intestinal dysbiosis is recognized as one of the pathogenic
factors for the development of several pancreatic diseases, such as
chronic alcoholic pancreatitis and pancreatic cancer [4, 5],
pathogenic roles of intestinal dysbiosis in AIP have remained poorly
defined. In our previous studies, we found that intestinal dysbiosis
mediates experimental AIP through the activation of pDCs, which
subsequently produce large amounts of IFN-α and IL-33 [20]. In this
study, we demonstrated differences in the composition of fecal
microbiota in samples from three type 1 AIP patients, at active and
remitted phases. Interestingly, induction of clinical remission by PSL
was accompanied by complete disappearance of Klebsiella spp. from
the gut of two patients who harbored this bacterium before the
treatment. We then explored the pathogenicity of Klebsiella spp.in experimental AIP by utilizing MRL/MpJ mice treated with 10 µg of poly
(I:C). In contrast to the manifestations in the well-established severe
AIP model induced by repeated IP injections of 100 µg of poly (I:C),
repeated IP injections of poly (I:C) at a tenfold lower dose (10 µg)
into MRL/MpJ mice resulted in the development of mild AIP [20]. By
utilizing this mild AIP model, we found that the degree of AIP was
greater in mice treated with both oral administration of heat-killedK. pneumoniae and IP injections of 10 µg of poly (I:C) than in
those that received either treatment alone. Moreover, oral
administration of heat-killed K. pneumoniae combined with IP
injections of poly (I:C) promoted pancreatic accumulation of pDCs;
consequently, this elevated IFN-α and IL-33 production by those cells.
Thus, moderate AIP mediated by the pancreatic accumulation of pDCs was
successfully induced in MRL/MpJ mice only when oral administration of
heat-killed K. pneumoniae was combined with IP injections of poly
(I:C). Therefore, oral administration of heat-killed K.
pneumoniae increased the sensitivity to poly (I:C)-induced experimental
AIP. Together with the complete disappearance of Klebsiella spp.from feces of type 1 AIP patients at the remission phase, these data
strongly suggest that immune responses against Klebsiella spp.may be involved in the development of type 1 AIP.
Induction of remission by PSL led to complete disappearance ofKlebsiella spp. from the feces of two of the three type 1 AIP
patients. In contrast, colonization by Ruminococcus spp. was
promoted after PSL treatment in all of the three patients. Thus, this
study suggests that gut colonization by Klebsiella spp. andRuminococcus spp. may play pro-inflammatory and anti-inflammatory
roles, respectively, in type 1 AIP. Such increased abundance ofKlebsiella spp. and decreased abundance of Ruminococcus
spp. have been observed in the feces of patients who developed
pancreatic fistula after pancreaticoduodenectomy for pancreatic cancer
[26]. In addition, fecal samples obtained from chronic alcoholic
pancreatitis patients exhibited higher abundance of Klebsiella
spp. than fecal samples from patients with alcoholic hepatitis
[27]. These reports and the present study support the view thatKlebsiella spp. may function as a pathobiont for CP and AIP as in
the case of primary sclerosing cholangitis (PSC) [28]. However, it
should be noted that our sample size was too small to establish with
certainty gut microbial alterations in type 1 AIP before and after PSL
treatment. Moreover, pathogenic immune responses to colonization byKlebsiella spp. might be masked by the differences in microbiota
profiles, as the proportions of Bacteroides, Streptococcus , andClostridium spp . were reported to be lower in patients with AIP
than in those with CP [29].
Oral administration of heat-killed K. pneumoniae increased the
sensitivity to experimental AIP induced by low doses of poly (I:C) in
this study. Thus, immune responses activated by heat-killed K.
pneumoniae alone were not sufficient for the induction of experimental
AIP. As for the pathogenic intestinal bacteria other than K.
pneumoniae , Haruta et al. reported that repeated IP injections of
heat-killed Escherichia coli led to the development of
experimental AIP in C57BL/6 mice [30]. They further identified FliC,
one of the flagella subunit proteins of this bacterium, as a bacterial
pathogenic factor because repeated IP injections of FliC alone caused
AIP in C57BL/6 mice [31]. Therefore, persistent exposure to FliC
derived from E. coli is sufficient for the development of
experimental AIP in C57BL/6 mice. These previous studies utilizing
heat-killed E. coli and C57BL/6 mice differed from our present
data in that the development of AIP in MRL/MpJ mice requires
administration not only of heat-killed K. pneumoniae but also of
poly (I:C) at low doses. Whether persistent exposure to heat-killed
intestinal bacteria plays a critical or supportive role in the
development of AIP remains to be elucidated. Nonetheless, those studies
and our present data indicate that the development of immune reactions
against intestinal bacteria may affect AIP.
One question arising from the present study is how heat-killed K.
pneumoniae activates pDCs, which produce large amounts of IFN-α and
IL-33. In this regard, host defense against pulmonary infection withK. pneumoniae depends upon TLR9-mediated proinflammatory cytokine
responses [32]. Moreover, the depletion of pDCs impairs
proinflammatory cytokine responses in mice during sublethal pulmonary
infection with K. pneumoniae [33]. Given the fact that TLR9
activation is a strong inducer of IFN-α production in pDCs [34, 35],
it is possible that heat-killed K. pneumoniae directly stimulates
IFN-α production by pDCs through TLR9 activation. This idea is supported
by the fact that activation of type I IFN signaling pathways, and
subsequent production of C-X-C motif chemokine ligand 10, mediate host
defense against murine K. pneumoniae pneumonia [36, 37].
Nakamoto et al. identified K. pneumoniae as one of gut
pathobionts for PSC [28]. Colonization by K. pneumoniaederived from PSC patients into gnotobiotic mice led to the development
of hepatobiliary injury through the induction of CD4+T cells, which produced IFN-γ and IL-17 [28]. Colonization byK. pneumoniae increased the sensitivity to experimental PSC, as
the development of chronic hepatobiliary inflammation required both
inoculation of this bacterium and oral administration of
3,5-dicarbethoxy-1,4-dihydrocollidine, the latter of which triggered
hepatobiliary injury. Together with our findings, those data suggest
that immune responses against K. pneumoniae may promote the
development of AIP and PSC in the presence of potent triggers. It should
be noted, however, that AIP and PSC are completely different in terms of
the sensitivity to PSL treatment in that most of patients with type 1
AIP respond to PSL treatment while those of PSC do not. Therefore, other
factors than the pathogenic colonization of Klebsiella spp. might
be involved in the different pathophysiology between these immune
disorders.
In conclusion, colonization by Klebsiella spp. may mediate the
development of type 1 AIP through the activation of pDCs.Klebsiella spp. may be a type 1 AIP pathobiont. However, a
confirmation of this hypothesis awaits future studies in which gut
microbiome will have to be studied in a large number of type 1 AIP
patients.