Cissus quadrangularis (Hadjod) Inhibits RANKL-Induced Osteoclastogenesis and Augments Bone Health in an Estrogen-Deficient Preclinical Model of Osteoporosis Via Modulating the Host Osteoimmune System.

Osteoporosis is a systemic skeletal disease characterised by low bone mineral density (BMD), degeneration of bone micro-architecture, and impaired bone strength. Cissus quadrangularis (CQ), popularly known as Hadjod (bone setter) in Hindi, is a traditional medicinal herb exhibiting osteoprotective potential in various bone diseases, especially osteoporosis and fractures. However, the cellular mechanisms underpinning its direct effect on bone health through altering the host immune system have never been elucidated. In the present study, we interrogated the osteoprotective and immunoporotic (the osteoprotective potential of CQ via modulating the host immune system) potential of CQ in preventing inflammatory bone loss under oestrogen-deficient conditions. The current study outlines the CQ's osteoprotective potential under both ex vivo and in vivo (ovariectomized) conditions. Our ex vivo data demonstrated that, in a dose-dependent manner CQ, suppresses the RANKL-induced osteoclastogenesis (p < 0.001) as well as inhibiting the osteoclast functional activity (p < 0.001) in mouse bone marrow cells (BMCs). Our in vivo µ-CT and flow cytometry data further showed that CQ administration improves bone health and preserves bone micro-architecture by markedly raising the proportion of anti-osteoclastogenic immune cells, such as Th1 (p < 0.05), Th2 (p < 0.05), Tregs (p < 0.05), and Bregs (p < 0.01), while concurrently lowering the osteoclastogenic Th17 cells in bone marrow, mesenteric lymph nodes, Peyer's patches, and spleen in comparison to the control group. Serum cytokine analysis further supported the osteoprotective and immunoporotic potential of CQ, showing a significant increase in the levels of anti-osteoclastogenic cytokines (p < 0.05) (IFN-γ, IL-4, and IL-10) and a concurrent decrease in the levels of osteoclastogenic cytokines (p < 0.05) (TNF-α, IL-6, and IL-17). In conclusion, our data for the first time delineates the novel cellular and immunological mechanism of the osteoprotective potential of CQ under postmenopausal osteoporotic conditions.

Long-term implications of COVID-19 on bone health: pathophysiology and therapeutics.

BACKGROUND: SARS-CoV-2 is a highly infectious respiratory virus associated with coronavirus disease (COVID-19). Discoveries in the field revealed that inflammatory conditions exert a negative impact on bone metabolism; however, only limited studies reported the consequences of SARS-CoV-2 infection on skeletal homeostasis. Inflammatory immune cells (T helper-Th17 cells and macrophages) and their signature cytokines such as interleukin (IL)-6, IL-17, and tumor necrosis factor-alpha (TNF-α) are the major contributors to the cytokine storm observed in COVID-19 disease. Our group along with others has proven that an enhanced population of both inflammatory innate (Dendritic cells-DCs, macrophages, etc.) and adaptive (Th1, Th17, etc.) immune cells, along with their signature cytokines (IL-17, TNF-α, IFN-γ, IL-6, etc.), are associated with various inflammatory bone loss conditions. Moreover, several pieces of evidence suggest that SARS-CoV-2 infects various organs of the body via angiotensin-converting enzyme 2 (ACE2) receptors including bone cells (osteoblasts-OBs and osteoclasts-OCs). This evidence thus clearly highlights both the direct and indirect impact of SARS-CoV-2 on the physiological bone remodeling process. Moreover, data from the previous SARS-CoV outbreak in 2002-2004 revealed the long-term negative impact (decreased bone mineral density-BMDs) of these infections on bone health. METHODOLOGY: We used the keywords "immunopathogenesis of SARS-CoV-2," "SARS-CoV-2 and bone cells," "factors influencing bone health and COVID-19," "GUT microbiota," and "COVID-19 and Bone health" to integrate the topics for making this review article by searching the following electronic databases: PubMed, Google Scholar, and Scopus. CONCLUSION: Current evidence and reports indicate the direct relation between SARS-CoV-2 infection and bone health and thus warrant future research in this field. It would be imperative to assess the post-COVID-19 fracture risk of SARS-CoV-2-infected individuals by simultaneously monitoring them for bone metabolism/biochemical markers. Importantly, several emerging research suggest that dysbiosis of the gut microbiota-GM (established role in inflammatory bone loss conditions) is further involved in the severity of COVID-19 disease. In the present review, we thus also highlight the importance of dietary interventions including probiotics (modulating dysbiotic GM) as an adjunct therapeutic alternative in the treatment and management of long-term consequences of COVID-19 on bone health.

COVID-19: Immunology, Immunopathogenesis and Potential Therapies.

The Coronavirus Disease-2019 (COVID-19) imposed public health emergency and affected millions of people around the globe. As of January 2021, 100 million confirmed cases of COVID-19 along with more than 2 million deaths were reported worldwide. SARS-CoV-2 infection causes excessive production of pro-inflammatory cytokines thereby leading to the development of "Cytokine Storm Syndrome." This condition results in uncontrollable inflammation that further imposes multiple-organ-failure eventually leading to death. SARS-CoV-2 induces unrestrained innate immune response and impairs adaptive immune responses thereby causing tissue damage. Thus, understanding the foremost features and evolution of innate and adaptive immunity to SARS-CoV-2 is crucial in anticipating COVID-19 outcomes and in developing effective strategies to control the viral spread. In the present review, we exhaustively discuss the sequential key immunological events that occur during SARS-CoV-2 infection and are involved in the immunopathogenesis of COVID-19. In addition to this, we also highlight various therapeutic options already in use such as immunosuppressive drugs, plasma therapy and intravenous immunoglobulins along with various novel potent therapeutic options that should be considered in managing COVID-19 infection such as traditional medicines and probiotics.

Elevated IL-6R on CD4+ T cells promotes IL-6 driven Th17 cell responses in patients with T1R leprosy reactions.

Th17 cells play vital role during pathogenesis of leprosy reactions. Previously, we have reported that IL-23 is involved in Th17 cells differentiation. Subsequently, our group also showed that IL-6 induces Th17 cell differentiation along with TGF-ß in leprosy reactions. Here, we next asked the question that whether IL-6 or IL-23 induced Th17 cells are different in nature? In this study, Type 1 Reactions (T1R) showed significantly (p < 0.001) higher percentage of IL-17A producing CD4+IL6R+ T cells as compared to non-reaction (NR) patients. Furthermore, recombinant IL-6, IL-23 and TGF-ß promoted IL-17A secretion by CD4+IL6R+ T cells. Subsequently, IL-6R and IL-23R blocking experiments showed significantly (p < 0.002) down regulated IL-17A in T1R reaction as compared to NR leprosy patients. The present study for the first time establishes that pathogenic Th17 cells produce IL-17 in an IL-6 dependent manner in leprosy T1R reactions. Thus, present approaches that specifically target Th17 cells and/or the cytokines that promote their development, such as IL-6, TGF-ß and IL-23A may provide more focused treatment strategies for the management of Mycobacterium leprae and its reactions.

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-ß and Increase in IL-6.

BACKGROUND: 50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions. METHODOLOGY AND PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-ß and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients. CONCLUSIONS: Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-ß and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.

CD4+ Th17 cells discriminate clinical types and constitute a third subset of non Th1, Non Th2 T cells in human leprosy.

BACKGROUND: Patients with localized tuberculoid and generalized lepromatous leprosy show respectively Th1 and Th2 cytokine profile. Additionally, other patients in both types of leprosy also show a non discriminating Th0 cytokine profile with both interferon-γ and IL-4. The present study investigated the role of Th17 cells which appear to be a distinct subtype of Th subtypes in 19 tuberculoid and 18 lepromatous leprosy patients. Five healthy subjects with long term exposure to infection and 4 skin biopsies from healthy subjects undergoing cosmetic surgery were used as controls. METHODOLOGY/PRINCIPLE FINDINGS: An array of Th17 related primers for cytokines, chemokines and transcription factors was used in real time reverse transcribed PCR to evaluate gene expression, ELISA for cytokine secretion in the supernatants of antigen stimulated PBMC cultures and flow cytometry for establishing the phenotype of the IL-17, IL-21 producing cells. CONCLUSIONS/SIGNIFICANCE: IL-17 isoforms showed significantly higher expression and release in supernatants of antigen stimulated PBMC cultures and dermal lesions of healthy contacts and tuberculoid leprosy as compared to lepromatous leprosy (p<0.003). This was further confirmed by Th17 associated transcription factor RORC, cytokines IL-21, IL-22, and IL-23, chemokines MMP13, CCL20, CCL22. Of interest was the association of IL-23R and not IL-6R with IL-17(+) cells. The Th17 cells were CD4(+) CCR6(+) confirming their effector cell lineage. Polarized Th1 cytokines were seen in 3/7 tuberculoid and Th2 cytokines in 5/10 lepromatous leprosy patients. Of importance was the higher association of Th17 pathway factors with the non-polarized Th0 types as compared to the polarized Th1 and Th2 (p<0.01). Our study draws attention to a third type of effector Th cell that may play a role in leprosy.

Lsr2 of Mycobacterium leprae and its synthetic peptides elicit restitution of T cell responses in erythema nodosum leprosum and reversal reactions in patients with lepromatous leprosy.

The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.