The respiratory tract, previously considered sterile, is one of the latest body sites being explored for the characterization of human-associated microbial communities. LM is a dynamic ecosystem whose composition in healthy lungs is likely to reflect microbial migration, elimination, and reproduction.

In detail, although certain bacteria are more abundantly represented in the airway microbiota than in the corresponding OM, primarily due to a selective advantage in replicating in the lung microenvironment compared to the oral one, a close resemblance of the LM to the OM has been documented [34]. The microbiome’s continuity in the lower respiratory tract is likely defined by the entry of bacteria into the lungs via regular OM microaspirations; conversely, LM members can propagate to the OM through coughing [35].

Moreover, it is now widely accepted that LM, intimately related to the GM, undergoes alterations in various respiratory disorders such as obstructive airway diseases [36], interstitial lung diseases [37], infections [38], and lung cancer [39]. Hence, we hypothesized a potential involvement of LM also in the pathogenesis of RCC/UCC.

Consequently, through a compositional and functional characterization of both intestinal and saliva microbiota of patients with RCC, we found, for the first time, that, compared with HC, patients presented a significant increase in microbiota alpha-diversity in saliva but not in stool samples. Significant differences in microbiota beta diversities were also observed between chronic coughers and HC in both intestinal and oral microbiota. Taken together, these findings support previous reports suggesting that gut and lungs are linked organs and changes in the GM community can influence the LM and vice versa [13, 40].

For instance, modification in newborns’ diet influences the composition of their LM while fecal transplantation in rats induces changes in their LM [41]. Moreover, the LM becomes enriched with gut bacteria after sepsis [42] and LPS instillation in the lungs of mice is associated with GM disturbances [43].

Consisted with our findings, a significant increase in oral alpha-diversity has been reported in patients with asthma [44] or COPD [45] compared with HC. However, no significant differences have been reported in fecal alpha-diversity indices among COPD patients and healthy subjects [46]. Moreover, a significant parting of the intestinal and saliva microbiota among RCC patients and HC has been documented.

Finally, in comparison with HC, RCC patients reported a significant increase in the fecal F/B ratio and a significant decrease in the saliva F/B ratio. In particular, an increased fecal F/B ratio has been associated with elevated lung IL-17 and IL-22 responses and enhanced airway hyperresponsiveness [47]. In general, these microbial compositional alterations in both oral and intestinal microbiota of RCC patients reflect the presence of a remarkable dysbiosis condition.

The analysis performed at all taxonomic ranks as in stool as in saliva samples also revealed significant differences in several taxa between RCC patients and HC. In particular, RCC patients reported higher fecal abundances of Erysipelotrichaceae family and Anaerostipes, Blautia., CAG-352, Enterorhabdus, and Streptococcus genera. In line with our findings, Erysipelotrichaceae members increased in COPD patients [45] but, in contrast to our results, Blautia, Anaerostipes, and Streptococcus genera were reduced in the GM of patients with cystic fibrosis or COPD [48, 49]. Moreover, we documented reduced abundances of bacteria belonging to Bacteroidota and Proteobacteria phyla, Gammaproteobacteria class, Enterobacterales order, Marinifilaceae family, and Acidaminococcus, Alloprevotella, Butyricimonas, Clostridia_vadinBB60_group, Mitsuokella, Odoribacter, Parabacteroides, and Sutterella genera in RCC patients compared to HC.

Regarding differences at the phylum level, Bacteroidota members are known to be overrepresented in healthy people [50] while Proteobacteria have been reported as relevant producers of lipopolysaccharide (LPS), which is in turn implicated in COPD development [51]. Notably, reduced levels of Enterobacteriaceae and Acidaminococcaceae have been reported in asthmatic patients [52, 53] while Lai et al. highlighted a significant negative association between Parabacteroides goldsteinii and COPD severity [54]. Additionally, Chiu et al., documented a lower abundance of Alloprevotella spp. in patients with rapid lung function decline [46] while a decreased abundance of Odoribacter spp. has been linked to different microbiota-associated diseases, such as inflammatory bowel disease and cystic fibrosis [55]. Finally, lower levels of Butyricimonas spp. have been associated with a detrimental decrease of butyric-acid production, a renowned SCFA with potent anti-inflammatory properties [56].

Concerning the saliva samples, RCC showed higher saliva levels of members of Spirochaetota phylum, Flavobacteriales order, Saccharimonadaceae and Selenomonadaceae families, and Capnocytophaga, Dialister, Porphyromonas, Saccharimonadaceae, and Selenomonas genera than HC.

Notably, Spirochaetota and Porphyromonas species have been widely associated with the pathogenesis of the periodontal disease [57, 58], a condition that may worsen COPD outcomes and play a causal role in the occurrence of pneumonia and bronchitis.

In contrast, Flavobacteriales, Capnocytophaga, Dialister, and Selenomonas species were significantly increased in the LM of COPD patients [45, 59].

In addition, our results showed reduced levels of bacteria belonging to Proteobacteria and Bacilli phyla, Gammaproteobacteria class, Micrococcales order, Burkholderiaceae, Micrococcaceae and Pasteurellaceae families, and Alloprevotella, Lautropia, and Rothia genera in RCC compared to HC.

In line with these findings, the relative abundance of Gammaproteobacteria, Bacilli, and Micrococcaceae members were decreased in asthmatic patients [60, 61] while lower levels of Rothia mucilaginosa, a common bacteria having inhibitory effects on pathogen- or LPS-induced pro-inflammatory responses, have been reported in patients with chronic lung disease [62].

Furthermore, to better characterize the consequences of these changes in both intestinal and oral microbiota, we performed a predictive functional analysis using the PICRUSt2 software. In detail, compared to HC, RCC patients showed a potential upregulation in the fecal pathways of lipopolysaccharide biosynthesis and lipoic acid metabolism but a lower biosynthesis of ansamycins, which conversely resulted potentially upregulated in saliva samples of RCC patients.

LPS is among the most potent microbial inducers of inflammation and is implicated in the deleterious effects of pulmonary infections. Animal models have reported that ML-7, a potent myosin light-chain kinase (MLCK) inhibitor, impedes neutrophilic inflammation caused by LPS in various respiratory diseases [63]. Interestingly, RCC patients exhibited a high metabolism of lipoic acid but its beneficial role in ameliorating many respiratory diseases (e.g., lung cancer, fibrosis, asthma, and acute lung injury) has been suggested because it shows anti-oxidative and anti-inflammatory properties [64].

On the other hand, ansamycins are secondary metabolites, mainly produced by Actinobacteria, known for their antimicrobial properties and currently used as the first-line treatment of tuberculosis [65]. Our results showed a high representation of Actinobacteria in both fecal and saliva microbiota of RCC patients, yet the potential upregulation of ansamycins biosynthesis was observed only in saliva samples but not in stool.

Finally, a microbial functional evaluation has been assessed through the analysis of fecal SCFA abundances and the evaluation of serum circulating FFAs in RCC patients and HC. About fecal SCFA, no statistically significant differences were found between RCC patients and HC, mainly because no SCFA-producing bacteria resulted differently abundant between groups. However, it’s noteworthy that a significant decrease in the total fecal content of SCFAs has been detected in some lung diseases including COPD and asthma [66, 67].

Anyway, regarding serum FFA abundances, RCC patients showed a significant increase in hexanoic acid, a bacterial metabolite known for its pro–inflammatory role through the activation of p38 MAPK signaling [68]. Moreover, in comparison to healthy subjects, RCC patients reported significantly reduced levels of various SCFA, MCFAs, and LCFAs.

Circulating FFA exerts well-established pleiotropic functions, ranging from maintaining an intestinal–epithelial integrity to dampening inflammation in the gut and respiratory tract [16]. While SCFAs promote the differentiation of immune-suppressive T regs in the gut [69], their detection in the lungs is limited, possibly due to the absence of digestible substrates [16]. However, Trompette et al. documented that, along the gut–lung axis, SCFAs play a protective role against allergic airway diseases and respiratory infection by priming myeloid cells in the bone marrow. These cells subsequently migrate to the lungs, shaping an anti-inflammatory milieu [70].

In RCC patients, we also documented a strong anti-correlation between saliva Dialister spp. abundance and serum levels of anti-inflammatory propionic and isobutyric acids. Dialister species are known intestinal SCFA producers [71] but an increased abundance of saliva Dialister spp. has been associated with oral and lung diseases [72, 73]. Importantly, Dialister spp. showed an anti-correlation with serum neutrophil to lymphocyte ratio and platelet lymphocyte ratio; two parameters increased in stable COPD patients [74].

Overall, we speculate that these alterations in intestinal and oral microbiota may play a role in RCC development through a complex cross-talk involving the gut, lungs, and brain. The bidirectional communication between the central and the intestinal nervous system, involving nerves, endocrine pathways, immunity, and microbial interactions, has been widely documented [75], with the bacterial SCFA acting as major metabolites that can affect various central nervous system (CNS) aspects [76].

In detail, SCFAs can directly or indirectly modulate vagal afferent fibers, leading to the activation of efferent fibers that conduct feedback signals from the CNS to the lungs, forming the “brain-lung axis.” This process promotes bronchial smooth muscle contraction, glandular secretion, mucosal swelling, and cough [77, 78]. Furthermore, intestinal and/or pulmonary dysbiosis can be the cause or contributory factor to a systemic and nervous hyperinflammatory state, which, in turn, disrupts both the intestine–brain and brain–lung communication pathways [78, 79].

This study has some limitations, including the restricted number of enrolled patients, and the evaluation of the LM composition only through saliva samples. However, we have documented, for the first time, numerous and consistent differences in the gut and oral microbial communities of RCC patients that could reflect an unbalanced gut–lung communication. Hence, although future studies are needed, these findings introduce new impacting factors in RCC pathogenesis, paving the way for further investigations and the development of novel therapeutic interventions for RCC management based on the modulation of microbial gut–lung communication.