Lipopolysaccharide （LPS）, a Gram-negative bacterial outer membrane component, is a representative immunostimulant, with terminal glycolipid lipid A as its active principle. Immunostimulant LPS is a potential candidate for adjuvants: immunomodulators that optimize vaccine efficacy. However, LPS is an endotoxin causing lethal sepsis. Thus, we need to minimize its toxicity before using it in vaccines. Focusing on parasitic and symbiotic bacterial LPS and lipid A, which are predicted to have low-toxicity immunomodulators as they have different LPS chemical structures due to co-evolution with the host, elucidation of the molecular basis of the parasitic and symbiotic phenomena has been progressed as well as the development of low-toxicity adjuvant has been conducted, based on the concept that there is a lipid A-mediated bacterial-host chemical ecology. In this review, recent developments in related research are presented.

Neonatal sepsis is an intractable disease with a high mortality, but the lack of an optimal animal model made it difficult to investigate its pathophysiology. We have established a non-surgical preterm sepsis mouse model by applying the cecal slurry method to 4-day-old newborn mice which equivalent to human preterm infants. In this paper, we will explain in detail the process of establishing the model.

Periodontitis is a chronic inflammatory disease in oral tissue caused by subgingival bacterial biofilms, including Aggregatibacter actinomycetemcomitans （A. actinomycetemcomitans）, and is also well known as a risk factor or trigger of autoimmune diseases （e.g., rheumatoid arthritis, ulcerative colitis, multiple sclerosis）. Moreover, Aggressive or juvenile periodontitis also has a strong relation with A. actinomycetemcomitans. Periodontitis causes the production of inflammatory cytokines, such as Interleukin （IL）-1 or TNFα. The production is significant to form the pathology of periodontitis. However, the mechanism of the cytokine production is unclear. Among inflammatory cytokines, IL-1 is produced by the particular signaling pathway because this cytokine lacks the signal sequence for spontaneous secretion. The expressed pro-IL-1 is proteolytically processed by activated caspase-1 and converted as mature cytokines. The caspase-1 activation is regulated by inflammasome （a complex of caspase-1）. Recent studies showed that caspase-11 recognizes lipopolysaccharide （LPS） from Gram-negative bacteria resulting in noncanonical Nod like receptor pyrin domain containing 3 （NLRP3） inflammasome activation with IL-1β production and pyroptotic cell death．

In this study, we provided that A. actinomycetemcomitans enhances progression of arthritis in mice model including cell infiltration and IL-1β production in the paws, and induces noncanonical inflammasome activation in macrophages in a caspase-11-dependent manner. We also found that invasion of A. actinomycetemcomitans into macrophages via integrin alpha M （CD11b） is necessary to activate the noncanonical inflammasome. Furthermore, we exhibited anti-CD11b antibody and caspase-11 deficiency inhibits progression of arthritis with attenuation of IL-1β production in the paws in mice model. These data elucidate molecular mechanisms underlying A. actinomycetemcomitans trigger noncanonical inflammasome activation and exacerbating rheumatoid arthritis.

It has been widely recognized that sepsis causes hypercytokinemia in the acute phase, needing to be abated as soon as possible. On the other hand, we reported that critically ill patients with extreme hypercytokinemia or with sky-high interleukin （IL）-6 blood levels show some specific genotypic distributions on upstream cytokine genes, e.g., TNF promoter single nucleotide polymorphism （SNP） and IL1RA variable number of tandem repeat （VNTR）. Thus, SIRS（systemic inflammatory response syndrome）was thought to precede CARS（compensatory anti-inflammatory response syndrome）, but recently, both are found to occur concurrently after the septic insult. CARS, or immunoparalysis, is one of the harmful host responses that is more challenging to address than SIRS. Precision immunotherapy garners attention as a counter-measure against the pathophysiology. Inhibitory receptors such as PD-1 and CTLA-4, immune checkpoint inhibitors, are representative compounds for immune-enhancing therapy. A PD1 SNP （rs11568821） was reported to be associated with the 90-day mortality in sepsis. Also, the two SNPs on anti-apoptotic BCL2 are related to the incidence rate of acute kidney injury secondary to infection. The recent randomized control trial for sepsis （SCARLET） is designed to target a specific patient cohort with coagulopathy and organ dysfunctions, meaning precision medicine. Intensivists should place emphasis on measures taking the genetic conditions of critically ill patients into consideration in the future.

To establish a sepsis model mouse with gradual onset of symptoms that preserves the intraperitoneal drug administration route, we have created a new mouse model of sepsis using subcutaneous cecal slurry administration or intraperitoneal administration of heat-inactivated cecal slurry. Our new methods induce sepsis more slowly compared to conventional intraperitoneal administration of cecal slurry.

Neonatal sepsis is characterized by systemic bacterial invasion followed by a massive inflammatory response. Until now, no therapeutic strategy has been established that significantly reduces the mortality from neonatal sepsis in clinical setting. Recently, we have reported the protective role of an initial low-dose septic challenge （4-days of age） in preventing subsequent lethal sepsis （7-days of age） in a neonatal mouse using cecal slurry （CS） model （Nakasone et al. JCM 2021）. In this further study, we found that the protective effect of an initial low-dose septic challenge against lethal sepsis works even with different CS stock solutions, and the effect lasts up to 2 weeks.

Gingipains, cysteine proteases produced by the periodontopathic bacterium Porphyromonas gingivalis, not only exacerbate periodontal disease but have also been implicated in the pathogenesis of Alzheimer’s disease （AD）, rheumatoid arthritis or diabetes mellitus. Neutrophil extracellular traps （NETs） released by oral neutrophils contribute to the maintenance of oral homeostasis, whereas NETs released upon stimulation by periodontopathic bacteria exacerbate inflammatory responses. This review outlines the impact of periodontal disease on the pathogenesis of brain dysfunction, including AD, focusing on gingipains and NETs. Gingipains have been implicated in the induction of chronic inflammation in periodontal disease. Gingipains have also been implicated in the pathogenesis of AD, and clinical trials of gingipain inhibitors have shown improved rates of cognitive decline in periodontal patients with P. gingivalis infection. Oral neutrophils play a role in infection defense by continuously releasing NETs upon stimulation by oral flora and saliva. However, NETs released by oral neutrophils upon stimulation by periodontopathic bacteria have lost their ability to protect against infection and induce inflammation.

The 14- and 15-membered macrolide antibiotics, such as erythromycin, clarithromycin （CAM）, and azithromycin, have immunomodulatory activity. Low-dose and long-term administration of the macrolides leads to symptom improvements of neutrophilic chronic inflammatory airway diseases, such as diffuse panbronchiolitis, chronic sinusitis, sinobronchial syndrome, cystic fibrosis, and chronic obstructive pulmonary disease. Suppression of proinflammatory cytokine and mucin production has been reported as a mechanism of the immunomodulating activity. Although the macrolides have beneficial immunomodulatory activity, there is a concern that the use of drugs other than antibacterial activity may lead to the development of resistant bacteria. Identifying target molecules is valuable and essential information for screening new immunomodulating drugs without antibacterial activity. We previously screened CAM-binding proteins in human airway respiratory cells. Mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal-associated protein 25-like （NIPSNAP）1 and 2 were identified. LPS-induced IL-8 production and mitochondria respiration/metabolism were suppressed by knockdown either NIPSNAP1 or 2 using siRNA and shRNA. Inhibition of mitochondria function by deferiprone or m-chlorophenylhydrazone reduced the LPS-induced IL-8 production. CAM inhibited mitochondria respiration and metabolism. Our observations indicate that mitochondrial proteins NIPSNAP1, 2 are a target molecule of CAM for immunomodulatory activity, and the immunomodulation links to mitochondria function, such as respiration and metabolism.

Skeletal muscle is the largest organ in the human body and has important roles, including in movement, amino acid storage, glucose uptake, and the secretion of myokines. Various types of insult, including sepsis, burns, and cancer, activate interleukin-6 （IL-6）/janus kinase （JAK） 2/signal transducer and activator of transcription （STAT） 3 signaling and induce skeletal muscle atrophy. The IL-6/JAK2/STAT3 pathway activates proteolytic processes, including the ubiquitin-proteasomal, autophagy, and apoptosis pathways；and inhibits pathways that upregulate protein synthesis, such as the insulin-like growth factor-1 pathway. IL-6/JAK2/STAT3 signaling also inhibits skeletal muscle myogenesis by reducing the activities of regulators of myogenesis. Because the skeletal muscle atrophy induced through these mechanisms adversely affects both the survival and functional outcomes of patients, there is a need to develop novel strategies that target IL-6/JAK2/STAT3 signaling, such as those discussed in this review, as a means of reducing skeletal muscle atrophy.

Recent study on the activation of the Limulus amebocyte lysate （LAL） revealed that the dimerization is necessary for the activation of Factor C on lipopolysaccharide （LPS） aggregates. Factor B is also an LPS-binding protein and is activated on the same LPS aggregates. This indicates that the activation of the LAL needs a space on endotoxin aggregates. Therefore, monomeric LPS cannot activate the LAL. It may be difficult to be monomeric for LPS in water-based solutions. In animal blood, however, monomeric LPS can exist because soluble CD14 （sCD14） can bind to monomeric LPS. The sCD14/LPS complex is probably not detectable by the LAL because it is monomeric. This indicates that there may be undetectable LPS in animal blood. Detection of the undetectable LPS in animal blood can be important to understand the effect of LPS on animals. Methods to detect the undetectable LPS should be developed in the future.

The severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2）-related proteins, angiotensin-converting enzyme 2 （ACE2） and transmembrane protease serine 2 （TMPRSS2）, which promote the entry of the virus into host cells, are determinants of SARS-CoV-2 infection. Although these proteins are expressed in oral-related tissues, their expression patterns and modulatory mechanisms in the salivary glands remain unknown. We herein showed that full-length ACE2, which has both a fully functional enzyme catalytic site and high affinity SARS-CoV-2 spike S1-binding sites, was more highly expressed in salivary glands than in oral mucosal epithelial cells and the lungs. Regarding TMPRSS2, zymogen and the cleaved form were both expressed in the salivary glands, whereas only zymogen was expressed in murine lacrimal glands and the lungs. Metformin, an adenosine monophosphate-activated protein kinase activator, increased stimulated saliva secretion and full-length ACE2 expression and decreased cleaved TMPRSS2 expression in the salivary glands, and exerted the same effects on soluble ACE2 （sACE2） and sTMPRSS2 in saliva. Collectively, these results suggest that the protein expression patterns of ACE2 and TMPRSS2 in the salivary glands differ from those in other oral-related cells and tissues, and also that metformin affect the salivary expression of ACE2 and TMPRSS2, which have potential as targets for preventing the transmission of SARS-CoV-2.

Studies on systemic lupus erythematosus （SLE） model mice and human SLE patients have revealed that Toll-like receptor （TLR） 7 is involved in the onset of SLE. Since systemic autoimmune diseases develop due to enhanced expression of TLR7, we predicted that the TLR7 response is particularly tightly regulated, and we identified molecules associated with TLR7 using liquid chromatography-mass spectrometry. The molecule we identified was a small G protein called Arl8b, which is localized in endosomes and lysosomes. Arl8b is involved in the movement and fusion of endosomes and lysosomes, and regulates type 1 interferon production induced by TLR7 stimulation. Since the constitutive type 1 interferon production induced by TLR7 stimulation may be closely linked to the onset of SLE, we created BXSB.Yaa Arl8b-deficient mice by crossing BXSB.Yaa mice, which develop SLE in a TLR7-dependent manner, with Arl8b-deficient mice. The BXSB.Yaa Arl8b-deficient mice did not develop SLE at all. Furthermore, in a model of SLE induction by pristane administration, in which TLR7-dependent type 1 interferon causes SLE, the disease induction was significantly suppressed in Arl8b-deficient mice. Our research to date has revealed that Arl8b is involved in the onset of SLE through TLR7-dependent and TLR7-independent pathways. Research on how Arl8b is involved in the pathogenesis of SLE is expected to make significant progress in the future.

Extracellular vesicles （EVs）, secreted in response to microbial infection, play a crucial role in modulating immune and infectious responses. Sepsis, a life-threatening multiple organ dys-function caused by a systemic dysregulated inflammatory response to infection, poses signif-icant challenges in therapeutic interventions. Despite extensive trials, achieving satisfactory therapeutic outcomes remain elusive. Our prior research unveiled the potential of LL-37, a human cathelicidin host-defense peptide, in improving the survival rates of septic mice subjected to cecal ligation and puncture （CLP）. We herein explored the capacity of LL-37 to modulate the functions of neutrophils, the primary defenders against pathogens, focusing on its ability to enhance the secretion of anti-inflammatory EVs and promote cell motility. In vitro stimulation of neutrophils with LL-37 led to elevated secretion of EVs （PMN-LL-37-EV） containing anti-microbial molecules such as lactoferrin and CRAMP, consistent with their observed antibac-terial activity. Injection of PMN-LL-37-EV into CLP mice significantly improved survival rates and reduced bacterial burdens. Notably, lung tissues from PMN-LL-37-EV-injected mice ex-hibited reduced inflammation. 3D imaging by a Lattice light-sheet microscopy with a time-lapse function revealed heightened neutrophil motility following LL-37 treatment. In addition, ad-ministration of LL-37 to CLP mice resulted in enhanced neutrophil infiltration and EV release at the site of inflammation, within the peritoneal cavity. Taken together, LL-37 orchestrates the motility of neutrophils to inflammatory sites and promotes the secretion of an-ti-bacterial/anti-inflammatory EVs in septic mice, thereby attenuating bacterial loads and con-ferring protection against lethal septic conditions.

Yokuinin （Coix lacryma-jobi var. ma-yuen seed extracts) is a traditional chinese medicine, which has been used for treatment of warts, chapped skin, rheumatism, and inflammatory diseases. Yokuinin has anti-inflammatory, anti-oxidative stress, anti-tumor, analgesic, blood sugar-lowering, and immunomodulatory effects. However, the molecular mechanism by which Yokuinin have immunomodulatory effects remains largely unknown. In this study, we demonstrated that Yokuinin regulated the production of cytokines in bone marrow derived -macrophages （BMDMs) and -dendritic cells （BMDCs).

We have analyzed the levels of TNF-α and IL-6 in the culture-media samples collected from non-treated and Yokuinin crude extracts （YCEs)-treated BMDMs and BMDCs. We found that YCEs induced TNF-α and IL-6 production were significantly increased in dose-dependent manner. Furthermore, Coix polysaccharides （CPS) could increase the production of TNF-α and IL-6 via NF-κB and MAP kinase pathway in macrophages. These results suggest that CPS is a potentially immunomodulatory for activating macrophages.